CN112057443B

CN112057443B - Medical application of benzene sulfonamide compound and pharmaceutical composition thereof

Info

Publication number: CN112057443B
Application number: CN202011070259.5A
Authority: CN
Inventors: 王琛; 洪泽; 余文颖; 孙宏斌; 梅家豪; 李晨辉; 刘星; 孙立; 柳军
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-10-12
Filing date: 2020-10-09
Publication date: 2022-10-14
Anticipated expiration: 2040-10-09
Also published as: CN112057443A; WO2021068951A1

Abstract

The invention discloses a medical application of benzene sulfonamide compounds and a pharmaceutical composition thereof, in particular to compounds shown as formulas I-V or pharmaceutically acceptable salts or solvates thereof, which can be used for preparing STING inhibitors or drugs for inhibiting the activation of STING signal pathways and drugs for preventing or treating STING-mediated diseases;

Description

Medical application of benzene sulfonamide compound and pharmaceutical composition thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to medical application of a benzene sulfonamide compound or pharmaceutically acceptable salt or solvate thereof and a pharmaceutical composition thereof, which can act on transmembrane protein 173 (TMEM 173), also called STING (stimulator of interferon genes), and inhibit a signal path thereof. Therefore, the compounds can be used for preparing medicaments for preventing or treating STING-mediated diseases.

Background

Activation of the body's innate immune system is mediated by pattern-recognition receptors (PRRs) expressed on cell membranes and in the cytoplasm that recognize non-self pathogen-associated molecular patterns (PAMPs). Upon recognition of PAMPs, PRRs activate the transduction of downstream immune signals, promote inflammation and the release of immune cytokines, further activate the adaptive immune system of the body to synergistically kill and eliminate invading pathogenic microorganisms (Cold Spring Harbor Symposia on Quantitative Biology,1989, 54. Similarly, the damage-associated molecular patterns (DAMPs) secreted by cells under abnormal conditions (stress, injury, senescence or death) are also recognized by pattern recognition receptors, which in turn activate the immune system and promote cellular regeneration and repair after injury (Science, 2002,296 (5566): 301-305).

DNA in the cytoplasm plays an important role as a classical PAMP or DAMP molecule in the activation of the innate immune system (Curr Opin Immunol,2018, 55. The cytoplasmic DNA receptor protein cGAS (Cyclic AMP-GMP synthsase) is capable of recognizing abnormally exposed cytoplasmic DNA molecules and catalytically synthesizing 2',3' -cGAMP molecules (2 '-3'/3'-5' Cyclic GMP-AMP) using ATP and GTP as substrates. 2',3' -cGAMP and the signal molecules c-di-AMP and c-di-GMP formed in bacterial metabolism are called Cyclic Dinucleotides (CDNs). These CDNs bind specifically to the endoplasmic reticulum regulatory protein STING (also known as TMEM173, MITA, ERIS or MPYS) dimer forming a "V" shaped pocket (Nature, 2008,455 (7213): 674-678, nature,2011,478 (7370): 515-518), which in turn induces the activation of the polymerization of the STING protein (Cell, 2013,154 (4): 748-62). The multimeric STING protein migrates from the endoplasmic reticulum to the golgi compartment and recruits the downstream kinase protein TBK1 and the transcription factor IRF3 during this process, TBK1 catalyzing phosphorylation of STING and IRF3 upon autophosphorylation activation (Nature, 2019,567 (7748): 394-398, nature,2019,569 (7758): 718-722. Phosphorylated IRF3 dimerizes further into the nucleus, promoting the expression of type I interferons (type I interferons) and their associated immune factors (interferon stimulated genes, ISGs). In addition, STING proteins can also activate NF- κ B signaling pathway by recruiting TBK1 and TRAF6 molecules, and promote the expression of inflammatory factors such as TNF- α and IL-6 (J Virol,2014,88 (10): 5328-41).

Although STING-mediated activation of innate immune signaling pathways plays an important role in the body's defense against the invasion of pathogenic microorganisms, continued STING pathway activation leads to the development and progression of a variety of autoimmune and inflammatory diseases (Nature Immunology,2017,18 (7): 716-724). AGS syndrome (Aicardi-Gouti res syndrome) is a rare autoimmune disease that is systemic and caused by mutations in the TREX1, RNASE H2, SAMHD1, ADAR or IFIH1 genes (Am J Med Genet A,2015,167A (2): 296-312). Studies in animal models found that mutations in the genes for the nucleic acid metabolizing enzymes TREX1, RNASE H2 and SAMHD1 lead to cytoplasmic DNA aggregation that in turn activates the cGAS-STING signaling pathway, and further that knocking out expression of STING proteins in diseased mice significantly improved disease progression (J Exp Med,2016,213 (3): 329-36 proc Natl Acad Sci U S a,2015,112 (42): E5699-705 nature,2018,557 (7703): 57-61. Mutations in the TREX1 gene have also been found in Systemic Lupus Erythematosus (SLE) patients, and high expression of type I interferon is observed in 50% to 60% of SLE patients (Nat Rev Rheumatotol, 2018,14 (4): 214-228). In patients with Bloom syndrome, mutations in the BLM protein are found to lead to the formation of micronuclei, which in turn activates the STING signaling pathway to induce high expression of IFNs and ISGs in the serum of the patients (J Exp Med,2019,216 (5): 1199-1213). SAVI (STING-associated vasculopathy with initiation in diabetes) disease is a systemic autoimmune disease induced by abnormal activation of STING proteins due to mutations in individual amino acids (N Engl J Med,2014,371 (6): 507-518). <xnotran> , , (Nat Commun,2014,5:5166), (Nature, 2018,553 (7689): 467-472), (Nature, 2017,550 (7676): 402-406), (Shock, 5754 zxft 5754 (5): 621-631), (Gastroenterology, 2018,154 (6): 1822-1835), (Gastroenterology, 2018,155 (6): 1971-1984;Proceedings of the National Academy of Sciences,2017,114 (46): 12196-12201), (Nature Communications, 3252 zxft 3252 (1)), (Cell Metabolism,2019,DOI:10.1016/j.cmet.2019.08.003), (Nature Medicine, 3532 zxft 3532 (12): 1481-1487;EMBO Molecular Medicine,2020,7;12 (4): e 11002), (Nature, 2018,561 (7722): 258-262), (Journal of Neuroscience, 3425 zxft 3425 (2): 424-446), (Cell, 2020, 183:1-14;Biochemical and Biophysical Research Communications,2019,517 (4): 741-748) (Journal of Clinical Investigation,2020,130 (6): 3124-3136;Proceedings of the National Academy of Sciences,2020,117 (27): 15989-15999), (Nature, 2020,585 (7823): 96-101) , </xnotran> And the inhibition of the activation of the STING signaling pathway can significantly improve the occurrence and development of the above diseases. In conclusion, the development of inhibitors targeting STING proteins has broad clinical application prospects.

The reported small molecule inhibitors of STING in the literature have low refractive index, weak activity and large side effects (Cell Reports 2018,25,3405-3421 acs med. Chem.lett,2019,10 (1), 92-97). Therefore, there is an urgent clinical need to develop novel small molecule inhibitors of STING.

Disclosure of Invention

The invention aims to: aiming at the problems in the prior art, the invention provides the application of the benzene sulfonamide compound shown in any one or more of the following formulas I to V or the pharmaceutically acceptable salt or solvate thereof in preparing the medicines for inhibiting the activation of the STING signal pathway and the application in preparing the medicines for preventing or treating the STING-mediated diseases. Through virtual screening, biological activity evaluation and action mechanism research, the invention discovers that the benzene sulfonamide compounds shown in the formulas I to V or pharmaceutically acceptable salts or solvates thereof can specifically inhibit the activation of the STING signal channel.

The invention also provides a pharmaceutical composition for preventing and treating STING-mediated diseases.

The technical scheme is as follows: in order to achieve the purpose, the invention discloses application of a benzene sulfonamide compound shown in any one or more of formulas I to V or a pharmaceutically acceptable salt or solvate thereof in preparing a medicament for inhibiting a STING signal pathway:

the invention relates to an application of one or more benzene sulfonamide compounds shown in formulas I-V or pharmaceutically acceptable salts or solvates thereof in preparing a medicament for preventing or treating STING mediated diseases.

Further, the STING-mediated disease comprises one or more of infectious diseases, inflammatory diseases, autoimmune diseases, organ fibrosis diseases, ischemic cardiovascular and cerebrovascular diseases, neurodegenerative diseases, brain trauma, spinal cord injury, cancer or cancer-stage syndromes.

In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, may be used for the prevention or treatment of infectious diseases, including: mycobacterium tuberculosis infection, chlamydia infection, herpes virus (herpes simplex virus) infection, adenovirus infection, hepatitis b virus infection, orthomyxovirus infection and coronavirus infection.

In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, may be used for the prevention or treatment of inflammatory diseases, including: metabolic inflammation-related diseases (such as insulin resistance, metabolic syndrome, type 1 or type 2 diabetes mellitus, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, ischemic encephalopathy, cerebral stroke, hemorrhagic encephalopathy, cerebral hemorrhage, cerebral edema, diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy and diabetic ulcer, non-alcoholic fatty liver, non-alcoholic steatohepatitis, alcoholic fatty liver, cirrhosis, gout, stroke or cerebral infarction, etc.), musculoskeletal muscle inflammation (hand, wrist, elbow, shoulder, neck, knee, ankle and foot joint inflammation, such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, etc.), eye inflammation (keratitis, scleritis, conjunctivitis, etc.), digestive system inflammation (colitis, hepatitis, cholangitis, cholecystitis, pancreatitis, gastritis, enteritis, inflammatory bowel disease, proctitis), nervous system inflammation (meningitis, neuromyotonia, multiple sclerosis, CNS vasculitis), degenerative nervous system disease (parkinson's disease, huntington's disease, familial amyotrophic lateral sclerosis), vascular or lymphatic system inflammation (vasculitis, lymphangitis, phlebitis), reproductive system inflammation (cervicitis, endometritis, epididymitis, orchitis, urethritis), respiratory system inflammation (pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, obliterative bronchiolitis, idiopathic pulmonary fibrosis, chronic bronchitis, chronic bronchitis, etc.), cystic fibrosis lung disease), organ fibrotic diseases (liver fibrosis, kidney fibrosis, lung fibrosis, myocardial fibrosis), other inflammatory conditions including appendicitis, myocarditis, mumps, gingivitis, prostatitis, peritonitis, pleuritis, vasculitis, phlebitis, edema, nephritis, spinal cord injury including trauma or other disease-induced spinal cord injury, brain trauma.

In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, may be used for the prevention or treatment of autoimmune diseases. The method comprises the following steps: aicardi-Gouti res syndrome (AGS), infant-onset STING-related vasculitis (SAVI), retinal vasculopathy with brain protein dystrophy (RCVL), systemic Lupus Erythematosus (SLE), familial lupus Chilblain (CHBL), behcet's disease, chagas ' disease, psoriasis, multiple sclerosis, scleroderma, behcet's disease, and the like.

In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, may be used to prevent or treat T cell mediated hypersensitivity reactions with inflammatory components, including urticaria, skin allergies, allergic rhinitis, contact dermatitis, respiratory allergies, and the like.

In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, may be used to prevent or treat cancer and tumor metastasis in various tissues and organs of the body, including but not limited to lung, bone, pancreas, liver, kidney, head, uterus, ovary, stomach, colon, esophagus, small intestine, endocrine system, prostate, bladder, cervix, vagina. Such as liver cancer, kidney cancer, cervical cancer, lung cancer, skin cancer, uterine cancer, adenocarcinoma, prostate cancer, sarcoma, osteosarcoma, thyroid cancer, non-small cell lung cancer, esophageal cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphocytic leukemia, multiple myeloma, malignant lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma.

In certain preferred embodiments, the STING-mediated disease is psoriasis, stroke, myocardial infarction, brain trauma, spinal cord injury induced by trauma or other disease, parkinson's disease, huntington's disease, familial amyotrophic lateral sclerosis, non-alcoholic steatohepatitis, systemic lupus erythematosus, aicardi-gouuti syndrome, rheumatoid arthritis, inflammatory bowel disease, STING-related vascular disease (SAVI) that occurs in infancy, or diabetes and complications thereof.

The pharmaceutically acceptable salts of the compounds shown in the formulas I to V are salts formed by metal ions or pharmaceutically acceptable amine, ammonium ions or choline.

The compounds of the present invention or pharmaceutically acceptable salts or solvates thereof may be used alone or in combination with other therapeutic agents. As an immunomodulator, the compounds of the present invention may be used in monotherapy or in combination with other therapeutic agents to treat diseases associated with dysfunction of STING proteins, including infectious diseases, inflammatory diseases, autoimmune diseases, organ fibrotic diseases, ischemic cardiovascular and cerebrovascular diseases, brain trauma, spinal cord injury, neurodegenerative diseases, cancer or precancerous syndromes.

The compounds of the present invention are useful as pharmaceutically acceptable salts. The salt may be an acid salt of at least one of the following acids: galactaric acid, D-glucuronic acid, glycerophosphoric acid, hippuric acid, isethionic acid, lactobionic acid, maleic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, pivalic acid, terephthalic acid, thiocyanic acid, cholic acid, n-dodecylsulfuric acid, benzenesulfonic acid, citric acid, D-glucose, glycolic acid, lactic acid, malic acid, malonic acid, mandelic acid, phosphoric acid, propionic acid, hydrochloric acid, sulfuric acid, tartaric acid, succinic acid, formic acid, hydroiodic acid, hydrobromic acid, methanesulfonic acid, nicotinic acid, nitric acid, orotic acid, oxalic acid, picric acid, L-pyroglutamic acid, saccharinic acid, salicylic acid, gentisic acid, p-toluenesulfonic acid, valeric acid, palmitic acid, sebacic acid, stearic acid, lauric acid, acetic acid, adipic acid, carbonic acid, benzenesulfonic acid, ethanedisulfonic acid, ethylsuccinic acid, fumaric acid, 3-hydroxynaphthalene-2-carboxylic acid, 1-hydroxynaphthalene-2-carboxylic acid, oleic acid, undecylenic acid, ascorbic acid, camphoric acid, ethanesulfonic acid. Alternatively, the salt may be a salt of a compound of the present invention with a metal ion (including sodium, potassium, calcium, etc.) or a pharmaceutically acceptable amine (including ethylenediamine, tromethamine, etc.), an ammonium ion or choline.

The present invention includes various deuterated forms of the compounds of the invention. Each available hydrogen atom attached to a carbon atom may be independently substituted with a deuterium atom.

The pharmaceutical composition for preventing or treating STING-mediated diseases comprises any one of benzene sulfonamide compounds shown in formulas I-V or pharmaceutically acceptable salts or solvent compounds thereof as an active ingredient and pharmaceutically acceptable auxiliary materials. The excipients which can be mixed arbitrarily may vary depending on the dosage form, administration form and the like. Examples of the auxiliary materials include excipients, binders, disintegrating agents, lubricants, flavoring agents, coloring agents, sweetening agents, and the like. The pharmaceutical composition can be in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches and other pharmaceutically conventional preparations.

The compounds of the present invention are commercially available from EnamineStore and can be prepared by the methods described in the examples or by modifications thereof. HPLC purity of all compounds was above 95%.

The invention screens compounds which can be combined with STING protein by a computer-aided virtual screening method, and further performs biological activity verification on the compounds at a cellular level. As a result, it was found that the compound of formula I (SN-011, CAS. For example, the half inhibition of IFN- β activation by the compound of formula I in mouse primary myelodifferentiated macrophages and human primary foreskin fibroblasts was around 100nM and 500nM, respectively. Further mechanism research finds that the compound shown in the formula I can obviously activate DNA-induced polymerization of STING protein in cytoplasm, inhibit the transfer of STING protein from endoplasmic reticulum to Golgi apparatus, inhibit the recruitment of downstream adaptor proteins TBK1 and IRF3, lead to the phosphorylation of transcription factors IRF3 and NF-kB and the obvious reduction of nuclear transcription level thereof, and further inhibit the expression of downstream type I interferon, IL-6 and TNF-alpha. In addition, the co-crystal structure of the human STING protein and the compound of the formula I is obtained and analyzed, and the result shows that a plurality of conserved amino acid sites in the STING protein have a binding effect with the compound of the formula I, including Tyr167, ser241, ser243, glu260 and Thr263. Notably, these sites are involved in the binding of STING proteins to their endogenous ligand molecule 2'3' -cGAMP. And further competitive Pulldown experiments confirm that the compound of formula I binds to the STING protein in a non-covalent, reversible manner. The surface ion resonance molecular interaction instrument (SPR) detected that the affinity between SN-011 and the human STING protein was 4.03nM, compared with the affinity between 2'3' -cGAMP and the STING protein being 9.23nM. This indicates that the compound of formula I has a stronger binding capacity to the STING protein than 2'3' -cGAMP.

Accordingly, the invention provides application of compounds shown in formulas I-V or pharmaceutically acceptable salts or solvates thereof in preparing medicines for inhibiting activation of STING signal pathways.

The invention selects a mouse Trex1 gene knockout autoimmune disease model, an SAVI in vitro cell model, a middle cerebral artery embolism (MCAO) induced rat and mouse stroke model, a high fat diet induced mouse non-alcoholic fatty liver model and an imiquimod induced mouse psoriasis model for pharmacodynamics evaluation of the compound. The results of the studies carried out with the compound of formula I as an example show that:

1. the compound in the formula I can obviously improve the inflammatory injury of multiple tissue organs of a Trex1 knockout mouse, inhibit the activation of an organism self-reaction adaptive immune system and prolong the long-term survival rate of a diseased mouse.

2. In cells over-expressed by SAVI-related STING point mutants, the compound of formula I can remarkably inhibit the expression of cell type I interferon and related ISGs and inflammatory cytokines thereof.

3. In a rat cerebral apoplexy model prepared by MCAO, the compound of the formula I can obviously reduce the expression of type I interferon and related ISGs and inflammatory cytokines in cerebral tissues of an ischemic area, and obviously improve the neurobehavioral function of cerebral apoplexy animals.

4. In a mouse stroke model prepared by MCAO, the compound of the formula I can be immediately administered to remarkably reduce the expression of inflammatory cytokines in brain tissues of an ischemic area and remarkably improve the memory function and the motor function of a mouse; the compound of the formula I can still remarkably improve the motor function of a stroke mouse after being administrated 24 hours after the stroke.

5. In a mouse non-alcoholic fatty liver model induced by long-term high-fat diet, the compound shown in the formula I can obviously improve the lipid aggregation and inflammatory infiltration of the liver of a mouse and improve the liver injury.

6. In establishing an imiquimod-induced psoriasis-like inflammation model in BALB/c mice, the compound of formula I can significantly improve psoriasis-like inflammation.

Based on the above research results, the present invention provides the use of compounds of formulae I-V or pharmaceutically acceptable salts or solvates thereof in the preparation of a medicament for the prevention or treatment of STING-mediated diseases.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) The invention discovers for the first time that any one of the benzene sulfonamide compounds shown in the formulas I-V or the pharmaceutically acceptable salt or solvate thereof can be used as a brand new type small molecule inhibitor for targeting STING, can inhibit the activation of a STING signal path with high efficiency, specificity and low toxicity, and can be used for preparing the medicine for inhibiting the activation of the STING signal path.

(2) The compounds of the invention act via a defined mechanism by inhibiting the activation of the STING signaling pathway by directly binding to the STING protein and maintaining its dimeric conformation in the resting state. Particularly, the compound of the present invention has significant in vivo efficacy in disease models of inflammatory diseases, autoimmune diseases, organ fibrosis diseases, ischemic cardiovascular and cerebrovascular diseases, etc., and thus, is expected to be used for preparing a medicament for preventing or treating the STING-mediated diseases.

Drawings

FIG. 1 is a diagram of the inhibitory effect of small molecule compounds containing a benzenesulfonamide structure on the STING signal pathway obtained by virtual screening and the structures of part of the compounds: (A) MEF cells are incubated with 10 mu M of test compound, after 6 hours of incubation, liposome transfection is carried out for 5 mu g of ISD stimulation for 6 hours, and the expression of cell detection Ifn beta gene is collected; (B) MEF cells are incubated with 10 mu M of test compound, and cells are collected after 6 hours of incubation to detect the expression of Ifn beta gene; (C) Chemical structures of compounds SN-001 to SN-004 and negative control compounds SN-100; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 2 is a graph of the effect of in vitro biological activity of compound SN-001 (compound of formula II): (A-D) after 5-20 mu M compounds SN-001 and SN-100 are respectively incubated for 6 hours on L929 cells, carrying out stimulation for 6 hours by transfecting HT-DNA 4 mu g with liposome, and collecting the expression of Ifn beta, ifn alpha 4, cxcl10 and Il6 genes detected by the cells; (E-H) incubating THP-1 cells for 6 hours respectively by 5-20 mu M compounds SN-001 and SN-100, stimulating for 6 hours by using 40 mu L of HSV-1 virus, and collecting cells to detect the expression of IFN beta, IFN alpha 4, CXCL10 and IL6 genes; (I) After 10 mu M compounds SN-001 and SN-100 are respectively incubated on L929 cells for 6 hours, the liposome is transfected with HT-DNA 4 mu g to stimulate for 4 hours, and the cells are collected to detect the expression level of corresponding protein by a specific antibody immunoblotting method of the protein; (J-K) after 10 μ M compounds SN-001 and SN-100 were incubated with L929 cells for 6 hours, 8 μ g of liposome-transfected HT-DNA was stimulated for 4 hours, immunofluorescence was labeled nuclear entry of transcription factors IRF3 and P65, nuclear location was indicated by DAPI staining, and the length of the ruler was 25 μ M; (L-N) after 5-20 mu M SN-001 compound is incubated in MEF, L929 and THP-1 cells respectively for 12 or 24 hours, the influence of SN-001 on the cell viability is detected by using an MTS reagent; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 3 is a graph of the in vitro biological activity effect of compound SN-011 (compound of formula I) and related compounds: (A) Respectively incubating L929 cells for 6 hours by 10 mu M SN-001 and related compounds SN-005-SN-011, transfecting HT-DNA by liposome for 4 mu g and stimulating for 6 hours, and collecting the expression of cell detection IFN-beta gene; (B) The compound structure of SN-001 and related compounds SN-005-SN-011; (C-E) 1 μ M Compound SN-011 after incubating MEF cells for 6 hours, respectively stimulating with 5 μ g of classical agonist ISD upstream of STING, 4 μ g of HT-DNA, 40 μ l of HSV-1, 1 μ g of C-di-GMP and 1 μ g of 2'3' -cGAMP, and detecting the expression of Ifn beta, cxcl10 and Il6 genes of the cells after the stimulation is completed; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 4 is the IC of Compound SN-011 (Compound of formula I) in MEF, BMDM and HFF cells ₅₀ Testing an effect graph: (A-C) incubation of gradient compound SN-011 (5 nM-5. Mu.M) in MEF, BMDM and HFF cells for 6 hours, stimulation with 2'3' -cGAMP 1. Mu.g for 3 hours, detection of IFN-beta gene expression after stimulation, mapping with inhibition rate of gene expression, and fitting IC ₅₀ A value; (D-F) after 1.25-20 mu M of compound SN-011 is respectively incubated in MEF, BMDM and HFF cells for 24 or 48 hours, the influence of SN-011 on the cell viability is detected by using an MTS reagent; n.s., no significant difference; * P is<0.05；**,P<0.01；

FIG. 5 is a graph of the effect of compound SN-011 (compound of formula I) specifically inhibiting the activation of the STING signaling pathway: (A-B) after 6 hours of incubation in STING knockout MEF cells for SN-011, separately transfecting CpG-DNA 5. Mu.g, poly (I: C) 5. Mu.g, sendai virus 10. Mu.l, IFN-. Beta.100U, or liposome with LPS 10. Mu.g, poly (I: C) 100. Mu.g, respectively, and detecting the expression of Ifn. Beta., il6, tnf. Alpha., isg15, and Isg56 genes after completion of the stimulation; (C) Respectively incubating the HFF cells for 1-10 mu M SN-011 for 12 or 24 hours, and respectively detecting the expression of cGAS, STING, TBK1 and IRF3 proteins after incubation is finished; (D-E) respectively incubating the HFF cells for 1-10 mu M SN-011 for 12 or 24 hours, and respectively detecting the expression of cGAS (MB 21D 1) genes and STING (TMEM 173) genes after incubation; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 6 is a graph of the effect of compound SN-011 (a compound of formula I) on inhibiting STING and its mediated activation of downstream signaling pathways: (A) After 1 mu M SN-011 or SN-100 is incubated on HFF cells overnight, 2'3' -cGAMP 1 mu g is used for stimulation for 1 hour, the cells are collected after the stimulation is finished, and phosphorylation and polymerization activation of STING protein are detected by an immunoblotting method; (B) After 1 mu M SN-011 or SN-100 incubates HFF cells overnight, 40 mu l HSV-1 virus infects the cells for 4 hours, the cells are collected after stimulation is finished, and the interaction of STING and the downstream proteins TBK1 and IRF3 is detected by a co-immunoprecipitation method; (C) After 1 mu M SN-011 or SN-100 incubate HFF cells overnight, using 2'3' -cGAMP 1 mu g to stimulate for 2 hours, collecting the cells, and detecting the expression level of the corresponding protein by using a specific antibody immunoblotting method of the protein; (D) After 1 mu M SN-011 or SN-100 is used for incubating HFF cells overnight, after 2'3' -cGAMP 1 mu g is used for stimulating for 2 hours, the cells are fixed, IRF3 protein entering nucleus is detected by an immunofluorescence method, the nuclear position is indicated by DAPI staining, and the length of a ruler is 25 mu M; (E) After 1 mu M SN-011 incubate Hela cells overnight, 40 mu l HSV-1 virus infects cells for 3 hours, after stimulation is completed, transfer of STING protein to Golgi apparatus is detected by immunofluorescence, the Golgi apparatus position is marked by GM130 staining, and the length of the ruler is 25 mu M;

FIG. 7 is a structural diagram of the co-crystal of compound SN-011 (compound of formula I) with hSTING-CTD (149-379) protein: (A) Schematic diagram of cocrystallization structure of SN-011 and hSTING-CTD (149-379) protein, two monomer molecules in dimer structure of STING protein are represented by green and blue, respectively, SN-011 molecule in pocket formed by dimer is represented by rod-shaped skeleton structure; (B) Schematic comparison of SN-011/hSTING-CTD (149-379) cocrystal structure (purple) with apo-hSTING-CTD (149-379) crystal structure (4 EMU) (yellow); (C) SN-011/hSTING-CTD (149-379) eutectic structure (purple) vs. 2'3' -cGAMP/hSTING-CTD (149-379) crystalline structure (4 LOH) (yellow); (D) The way in which the SN-011/hSTING-CTD (149-379) cocrystallization results are stacked in the crystal lattice; (E) Schematic interaction diagram of amino acid and small molecule on hSTING-CTD (149-379) protein after binding with SN-011;

FIG. 8 is a graph demonstrating the binding affinity and binding site of compound SN-011 (compound of formula I): (A-E) an SPR method is used for detecting the binding curves between small molecules SN-011, SN-100 and 2'3' -cGAMP and hSTING-CTD (149-379) protein, and simultaneously detecting the change of the intermolecular affinity between the protein and SN-011 after mutation of key mutation binding sites Ser241A, ser243A and Thr 263A; (F-G) transfecting 5 mu G of plasmids expressing Flag-hSTING and point mutants of Tyr167, ser241A, ser243A, glu260A and Thr263A into HEK293T cells respectively, adding SN-011 (10 mu M) to continue incubating for 12 hours after transfecting for 12 hours, collecting cells after incubating to detect the expression of IFN beta gene, and calculating the inhibition rate according to the gene expression value; (H-I) transfecting 5 mu g of plasmids expressing Flag-hSTING and point mutants of Tyr167, ser241A, ser243A, glu260A and Thr263A into HEK293T cells respectively, adding SN-011 (10 mu M) to continue incubating for 9 hours after transfecting for 12 hours, adding 2'3' -cGAMP 1 mu g to stimulate for 3 hours after incubating, collecting the expression of cells for detecting IFN beta genes, and calculating the inhibition rate according to the gene expression value; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 9 shows that compound SN-011 (compound of formula I) inhibits Trex1 ^-/- Effect profiles of highly expressed IFNs and ISGs in mouse primary BMDM cells: (A) Thermographic analysis SN-011 (500 nM) after 12 hours incubation Wild Type (WT) and Trex1 ^-/- RNA-Sequencing result of mouse BMDM cell gene expression; a total of 72 ISG genes are displayed on heatmaps, and two independent replicates were selected for each treatment group; (B-E) Trex1 ^-/- Incubation of DMSO, SN-0, in BMDM cells, respectively11 (500 nM) or SN-100 (500 nM), incubating for 12h overnight, and collecting cells to detect the expression of Ifn beta, cxcl10, isg15 and Il6 genes; calculation of Trex1 Using Gene expression of WT BMDM as a control group ^-/- Fold increase in expression of the gene of interest in BMDM cells; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P is<0.05；**,P<0.01；

FIG. 10 is a compound SN-011 (compound of formula I) mitigating Trex1 ^-/- Effect diagram of mouse multiple tissue organ inflammatory reaction: (A to D) WT and Trex1 ^-/- Injecting PBS or SN-011 (5 mg/kg) into abdominal cavity of mouse, injecting 3 times per week for 1 month, taking heart, stomach, tongue and muscle tissue of mouse, and detecting Ifn beta, cxcl10, isg15 and Il6 gene expression; (E) Taking the above tissue, fixing, and performing paraffin sectioning and H&E, staining, observing inflammatory cell infiltration of each tissue, counting data of at least 6 mice in each group, taking an average value and taking variance as an error line: n.s., no significant difference; * P is<0.05；**,P<0.01；

FIG. 11 is a compound SN-011 (compound of formula I) ameliorating Trex1 ^-/- Effect profile of systemic autoimmune response symptoms in mice: (A) WT and Trex1 were taken 1 month after SN-011 (5 mg/kg) injection ^-/- Spleen of mouse, after spleen cells were isolated, flow cytometry was used to detect activated T cells (CD 4) in spleen ⁺ CD69 ⁺ ,CD8 ⁺ CD69 ⁺ ) And memory T cells (CD 4) ⁺ CD44 ^high CD62L ^low ,CD8 ⁺ CD44 ^high CD62L ^low ) The content of (a); (B) Statistical analysis of T cell content of different subtypes in spleens of experimental mice; (C) WT and Trex1 months after injection of SN-011 (5 mg/kg) ^-/- Statistical analysis of survival of mice; (D) Taking WT and Trex1 after SN-011 injection for 1 month ^-/- The serum of the mouse is used for detecting the content of the antinuclear antibody in the serum of the mouse by an antinuclear antibody detection kit immunofluorescence method, the data of at least 6 mice in each group are counted, and the average value and the variance are taken as error lines in the experiment: n.s., no significant difference; * P is a radical of hydrogen<0.05；**,P<0.01；

FIG. 12 is a graph of the pharmacokinetic evaluation effect of compound SN-011 (compound of formula I) in mice: (A) Drug concentration in plasma of C57BL6 mice within 24 hours after a single intraperitoneal injection of SN-011 (5 mg/kg); (B) A pharmacokinetic parameter statistical table, wherein the blood concentration of 3 mice is taken for statistics in each time point;

FIG. 13 is a graph of the effect of toxicological assessments of compound SN-011 (compound of formula I) in mice: (A) WT mice were injected with PBS or SN-011 (1 mg/kg) intraperitoneally, and the body weight statistical analysis chart was obtained after 10 weeks of 3 consecutive injections per week; (B) Statistical analysis of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum of the mice; (C) Statistical analysis chart of Blood Urea Nitrogen (BUN) and serum Creatinine (CREA) in the serum of the mouse; (D) The heart, the kidney, the stomach and the liver of the mouse are respectively taken from the mouse, paraffin section and H & E staining are carried out after the fixation, the pathological change of each tissue is observed, each group at least counts the data of 6 mice, and the average value is taken in the experiment and taken as the variance as an error line: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 14 is a graph of the effect of compound SN-011 (compound of formula I) on inhibiting SAVI-associated STING point mutant-induced inflammatory gene expression: (A-C) transfecting and expressing Flag-hSTING and SAVI related point mutants V155M, N154S, G166E, C206Y, R281Q and R284G in HEK293T cells by 5 mu G of each plasmid, incubating a compound SN-011 (10 mu M) after 12 hours of transfection, and collecting cells after 12 hours of incubation of the compound to detect the expression of IFN-beta, CXCL10 and TNF-alpha genes; (D) Transfecting plasmids expressing Flag-hSTING and SAVI related point mutants of V155M, G166E, C206Y, R281Q and R284G in HEK293T cells by 5 mu G respectively, incubating a compound SN-011 (10 mu M) after transfecting for 6 hours, harvesting the cells after incubating the compound for 12 hours, and detecting recruitment of the STING to downstream target proteins TBK1 and IRF3 by using a co-immunoprecipitation method; (E) Transfecting plasmids expressing Flag-hSTING and SAVI related point mutants of V155M, G166E, C206Y, R281Q and R284G in HEK293T cells by 5 mu G respectively, incubating a compound SN-011 (10 mu M) after transfecting for 6 hours, harvesting the cells after incubating the compound for 12 hours, and detecting phosphorylation expression of TBK1 and IRF3 proteins by an immunoblotting method; (F) Plasmids expressing Flag-hSTING and SAVI related point mutants of V155M, G166E, C206Y, R281Q and R284G are transfected into HEK293T cells by 5 mu G respectively, after transfection for 6 hours, compounds SN-011 (10 mu M) are incubated, after compounds are incubated for 12 hours, cells are collected, and STING multimerization expression is detected by a method of non-denaturing gel electrophoresis; all data were averaged in triplicate and the variance was taken as the error bar: n.s., no significant difference; * P <0.05; * P <0.01;

FIG. 15 is a graph of the effect of compound SN-011 (a compound of formula I) on ameliorating acute cerebral ischemia-induced brain injury in rats: (A) After rat acute cerebral ischemia is induced by Middle Cerebral Artery Occlusion (MCAO) for 24 hours, detecting that an infarct area normal area of the brain is red and an infarct area is white by adopting a TCC (cross-staining) method; (B-C) scoring statistics is carried out on the ethology of the rat respectively for 6 hours and 24 hours after the middle cerebral artery of the rat is blocked; (D-I) after the middle cerebral artery is blocked and induces the acute cerebral ischemia of the rat for 24 hours, taking the tissue of the cerebral cortical area to detect the expression of Ifn beta, ifn alpha 4, cxcl10, mcp1, tnf alpha and Il6 genes; carrying out intraperitoneal injection on rats with low dose SN-011 (1 mg/kg) and high dose SN-011 (3 mg/kg) during and 12 hours after model building, carrying out statistical analysis on experimental results of 4 rats in each group, wherein n.s. has no significant difference; * P <0.05; * P <0.01, dosing group compared to MCAO group;

FIG. 16 Compound SN-011 (Compound of formula I) is a graph ameliorating the effects of acute cerebral ischemia-induced brain injury in mice: (A) Injecting low-dose SN-011 (1 mg/kg) and high-dose SN-011 (2 mg/kg) into the abdominal cavity immediately after the middle cerebral artery of the mouse is blocked, and detecting the expression of Mcp-1, il6 and Tnf alpha genes by taking cerebral cortex region tissues 24 hours later; (B) Injecting low-dose SN-011 (1 mg/kg) and high-dose SN-011 (2 mg/kg) into abdominal cavity immediately after the middle cerebral artery of the mouse is blocked, and detecting the expressions of Mcp-1, il6 and Cxcl10 genes by using cerebral cortex regional tissues after 72 hours; (C) Injecting SN-011 (2 mg/kg) into abdominal cavity immediately after or 24 hours after the middle cerebral artery of the mouse is blocked, continuously administering to the seventh day after the stroke, and determining the change of the motor function of the mouse by using a rotating rod experiment; (D) SN-011 (2 mg/kg) was administered intraperitoneally immediately or 24 hours after the middle cerebral artery of the mice was occluded, continuously until the seventh day after stroke, and the change in memory function of the mice was measured using the Morris water maze test. Statistical analysis is carried out on the experimental results of not less than 5 mice in each group, and no significant difference exists n.s.; * P <0.05; * P <0.01, administered group compared to Vehicle group after MCAO;

figure 17 is a graph of the effect of compound SN-011 (a compound of formula I) on improving the progression of High Fat Diet (HFD) -induced nonalcoholic fatty liver disease in mice: (A-B) mouse NAFLD model induced by high fat diet and ALT and AST content change in serum of each group of mice after administration; (C-D) the content of TC and TG in serum of each group of mice; (E) changes in body weight of mice in each group; (F) changes in liver weight in each group of mice; (G) Changes in visceral fat weight (perirenal fat) in each group of mice; (H-I) the contents of TC and TG in the livers of each group of mice; (J-O) changes in Ifn β, cxcl10, mcp-1, tnf α, fas and Srebp-1c gene expression in the livers of mice in each group; (P) analyzing H & E staining results of pathological liver sections of each group of mice; c57BL6 mice are induced by high fat diet for 20 weeks, and are intervened by injecting low-dose SN-011 (1 mg/kg) and high-dose SN-011 (2 mg/kg) into the abdominal cavity from the 10 th week, the experimental results of at least 8 mice in each group are statistically analyzed, and n.s. have no significant difference; * P <0.05; * P <0.01, dosing groups compared to HFD group;

FIG. 18 is a graph of the effect of Compound SN-011 (Compound of formula I) on the right ear and back of imiquimod-induced psoriasis-like inflammation mice: appearance of mice in a control group, a model group, an SN-011 250mg/kg group and an SN-011 50mg/kg group;

figure 19 is a graph of the effect of compound SN-011 (compound of formula I) on imiquimod-induced psoriatic-like inflammatory mouse ear thickness: mice in control group, model group, SN-011 250mg/kg group and SN-011 50mg/kg group had right ear thickness, P <0.05, <0.01, <0.001, = n 8;

FIG. 20 is a graph of the effect of compound SN-011 (compound of formula I) on the right ears and back of mice with imiquimod-induced psoriatic inflammation: A. h & E staining of mouse ear skin; B. h & E staining profile of skin on the back of mice, scale bar 50 μm (x 200); C. h & E staining is carried out on ear skins of the mice, the thickness of acanthopanax skin is analyzed, and the scale bar is 20 mu m (x 400); D. the dorsal skin of the mice was H & E stained and analyzed for spinous skin thickness with a scale bar of 20 μm (x 400), <0.05, <0.01, <0.001.N =8;

FIG. 21 is a schematic diagram of the mechanism of action of compound SN-011 (compound of formula I) in inhibiting the activation of the STING signaling pathway.

Detailed Description

The invention is further illustrated by the following figures and examples. The present invention is not limited by these examples. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention.

1. Chemical synthesis reagent and material

The raw materials and equipment used in the embodiment of the present invention are known products and commercially available.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or (and) Mass Spectrometry (MS). NMR was measured using a (Bruker) nuclear magnetic spectrometer using deuterated dimethyl sulfoxide (DMSO-d) as the solvent ₆ ) Deuterated chloroform (CDCl) ₃ ) Internal standard is Tetramethylsilane (TMS).

The column chromatography generally uses 200-300 mesh silica gel as carrier.

Known starting materials of the present invention may be synthesized by methods known in the art or may be purchased from Le Yan, available from bekka, alatin, an Naiji, and the like.

2. Cells and cell culture

The cell lines used by the invention comprise human kidney germ cells HEK293T, mouse embryo fibroblast MEF and L929, human cervical carcinoma cells Hela, human primary foreskin fibroblast HFF and the like. All cells were cultured using DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 50U/mL penicillin and 50. Mu.g/mL streptomycin (Gibco) and L929 using 1640 medium, except where otherwise indicated. Bone marrow-derived macrophage BMDM was derived from mouse femoral hematopoietic stem cells by differentiation. The differentiation time is generally 7 days, and the culture is carried out by using a conditioned medium containing L929 supernatant, and the corresponding stimulation can be carried out after the differentiation is finished.

3. Mouse strain and in vivo pharmacodynamic, pharmacokinetic and toxicology evaluations

C57BL/6J mice at 6-8 weeks were purchased from the University of Nanjing institute for model animals, and Trex1 heterozygous mice were offered by Dr. Homozygous knockout mice are obtained by crossing heterozygous mice (Cell Reports 2018,25,3405-3421). BALB/c mice (for anti-psoriasis efficacy test), female, aged 56-62 days in the day, were provided by Wentonlihua laboratory animals of Zhejiang, inc. In the experiment, rats and mice are raised in SPF animal rooms of the drug safety evaluation center of the Chinese university of pharmacy. Animal experiments were performed strictly in accordance with the operating guidelines established by the animal care committee of the university of chinese pharmacy.

In Trex1 ^-/- In the spontaneous autoimmune disease model of mice, mice were injected intraperitoneally with test compound SN-011 starting at week 4 at a dose of 5mg/kg mouse body weight, the compound dissolution was promoted with 2% final concentration of Tween20 and 2% DMSO, the solution was PBS, three times a week, and the administration was continued for 30 days.

Evaluation of SN-011 in C57BL6J mice in vivo pharmacokinetic experiments, intraperitoneal injection of 5mg/kg SN-011, 0.083,0.25,0.5,1,2,4,8,24 hours after plasma for blood concentration detection.

In a toxicological evaluation experiment of the compound SN-011 in a mouse, C57BL6J mouse is injected with 1mg/kg SN-011 intraperitoneally three times a week for 10 weeks continuously, and the biochemical indexes of the mouse serum and the pathological changes of tissues and organs are detected after the injection is finished.

In a model of acute cerebral ischemia induced by arterial occlusion in rats, male SD rats weighing 280-300g were administered the compound SN-011 at a low dose of 1mg/kg and at a high dose of 3mg/kg before and 12 hours after surgery, respectively. And detecting related indexes 24 hours after the operation. The rats in the experimental group were evaluated for behavioral scoring at 6 hours and 24 hours after the operation, respectively. The scoring rules are as follows: the grasping tail is bent only to the left side by 1 minute. The tail is caught on the ground and only turns for 2 minutes to the left side, and the tail is released and only turns for 3 minutes to the left side. Left hemiplegia was divided into 4.

In the acute cerebral ischemia model induced by artery occlusion in mice, male C57/6J mice weighing 25-30g were administered with compound SN-011 immediately after surgery or 24 hours after surgery, 1 time per day for the end of the experiment, with a low dose of 1mg/kg and a high dose of 2mg/kg.

In a High Fat Diet (HFD) -induced mouse non-alcoholic fatty liver disease (NAFLD) model, male C57BL6J mice of 4-6 weeks of age were fed High Fat diet (research Diets,60% kcal High-Fat Diets, D12492). After feeding with high-fat feed for 10 weeks, intraperitoneal injection of compound SN-011 was started, with a low dose of 1mg/kg and a high dose of 2mg/kg. Three times weekly dosing, 10 weeks consecutively, model groups were given 2% Tween20 and 2% DMSO in PBS. The relevant indicators were tested after the administration at week 20.

In pharmacodynamic experiments of the compound for improving imiquimod-induced psoriasis-like inflammation, 32 BALB/c mice, female and 56-62 days old in days are used. The feed is raised in a common environment, and the feed can freely eat drinking water. Preparation of compound SN-011 medicinal ointment: 250mg/kg group: 1) 560mg of the compound SN-011 are dissolved in 1ml of DMSO; 2) Adding lanolin (11.2 g) and vaseline (11.2 g) into 1mL diethyl ether, and heating to dissolve at 34 deg.C; 3) Adding the solution of 1) into the mixture of 2), adding 3ml of ethanol, stirring uniformly, and heating for a plurality of minutes; 4) Adding 0.05g of methyl paraben and 0.02g of propyl paraben dissolved in 0.4ml of diethyl ether into the mixture of 3), brushing with 200l of diethyl ether for 1-2 times, and adding into the mixture; 5) Heating the mixture of 4) at 88 deg.C for 60 min, and evaporating ethanol and diethyl ether to obtain ointment. 50mg/kg group: 1) 112mg of the compound SN-011 are dissolved in 500l of DMSO; 2) Adding lanolin (11.2 g) and vaseline (11.2 g) into 1mL diethyl ether, and heating to dissolve at 34 deg.C; 3) Adding the solution of 1) into the mixture of 2), adding 3ml of ethanol, stirring uniformly, and heating for a plurality of minutes; 4) Adding 0.05g of methyl paraben and 0.02g of propyl paraben dissolved in 0.4ml of diethyl ether into the mixture of 3), brushing with 200l of diethyl ether for 1-2 times, and then adding into the mixture; 5) Heating the mixture of 4) at 88 deg.C for 60 min, and removing ethanol and diethyl ether by evaporation to obtain ointment. 32 BALB/c mice were randomly divided by body weight into a blank Control group (Control group), a Model Control group (Model group), an SN-011 mg/kg group and an SN-011 50mg/kg group of 8 mice each, and back hairs were removed with a shaver to expose a skin area of 2cm X3 cm. 5% Imiquimod (IMQ) cream (topical dose 62.5 mg) was administered daily to the right ear and back. The drug is administered on the first to fifth days of the experiment, in the afternoon of the morning, on the sixth to seventh days of the experiment, in the afternoon of the morning, and in the noon, the drug is administered, in which the injection is performed, and the experimental period is 7 days. Starting from the imiquimod cream application, the mice were photographed daily and observed for the right ear and back conditions. The thickness of the right ear of each mouse was measured daily. The skin specimens of the back and the right ear of the mouse are soaked in 4 percent paraformaldehyde and embedded in paraffin. Staining with hematoxylin and eosin (H & E). The psoriasis skin lesion area, the disease severity (PASI) score, the right ear thickness and the like are subjected to data processing by adopting graphpad prism 5 software, and the multi-group is subjected to One-Way ANOVA (One-Way ANOVA).

4. Mouse tissue sections and associated pathology evaluation

Mouse tissue sections were taken and assessed for tissue damage and inflammatory cell infiltration by H & E staining. The specific experimental steps are as follows: 1) After completion of the administration period of the mice, each organ tissue of the mice was taken and fixed overnight with 4% pfa for standby; 2) Material taking: correspondingly trimming the cross section of the fixed tissue according to the requirement of an observation part, putting the tissue into an embedding frame, and performing gradient dehydration; 3) After dehydration, embedding the tissue with paraffin, and preserving the paraffin blocks of the tissue sample; 4) Trimming: after a complete tissue section is trimmed, the tissue section can be sliced, and the thickness of the slice is 5 mu m; 5) Placing the slices on water for spreading, taking out the slices by using a glass slide, and drying the slices at 56 ℃ to dissolve paraffin on the samples; 6) H & E dyeing, gradient dehydration, mounting and storing are carried out after gradient rehydration; 7) Observing the pathological change of the tissue under an upright microscope, and taking a picture and recording.

5. Detection of serum biochemical indicators

The biochemical indexes of the mouse serum comprise ALT, AST, TC, TG, GLU, BUN and CREA, and the detection of the indexes is carried out by using a division X-Panel plus biochemical analyzer in the medicine safety evaluation center of Chinese pharmaceutical university.

6. Detection of mouse liver Triglyceride (TG) and Total Cholesterol (TC) contents

The contents of TC and TG in the liver of the mouse were determined using a TG content detection kit (solarbio, BC 0625) and a TC content detection kit (solarbio, BC 1985) according to the instructions.

7. Detection of spleen T cell content

Detection of SN-011 pair Trex1 by flow cytometry ^-/- CD4 in mouse spleen ⁺ And CD8 ⁺ Influence of T cell content. The experimental procedure was as follows: 1) After the spleen cells pass through a 200-mesh filter screen, washing the cells on the filter screen into a centrifugal tube by using 10ml of PBS, re-suspending the cells, and centrifuging at 1200rpm for 5 minutes; 2) Lysing the cells with 4ml of red blood cell lysate; 3) Blocking, 20. Mu.l of Fc blocking per tube, blocking at 4 ℃ for 10 minutes, centrifugation at 1200rpm, washing once with PBS, and resuspending the cells in PBSA (1% BSA in PBS); 4) Antibody staining, antibody 1 diluted into PBSA and mixed with cells after mixing, antibody mix for T memory cells (CD 4-FITC, CD8-PE, CD44-APC, CD62L-Brilliant Violet 421), antibody mix for T activator cells (CD 4-FITC, CD8-PE, CD 69-APC); 5) The antibody was added and mixed well, incubated at 4 ℃ in the dark for 30 minutes, washed once with PBS, and resuspended in 500. Mu.l PBS to detect the corresponding T cell subtype content.

8. Detection of autoimmune antibodies in mouse serum

The method for detecting serum antinuclear antibodies (ANA) by using the ANA (HEp-2) antigen substrate slide kit (MBL-BION) detection kit is as follows: 1) Taking out the matrix sheet full of HEp-2 cells from the kit, and balancing at room temperature; 2) Serum was diluted with 2% bsa in PBS 1 and HEp-2 cells were incubated therewith, reacted for 30 minutes at room temperature and then serum was washed off with PBS; 3) Sucking water around the pore diameter by using absorbent paper, adding FITC (fluorescein isothiocyanate) -mouse IgG (immunoglobulin G) secondary antibody, reacting at room temperature for 30 minutes, and washing the secondary antibody by using PBS (phosphate buffer solution); 4) Mounting with Dako mounting reagent for preventing fluorescence quenching, and performing microscopic examination.

9. Molecular docking method for screening small molecule compounds binding with STING protein

The hSTING-CTD crystal structure PDB:4EF5 is used for molecular docking screening, the molecular docking software uses DOCK3.7, and the virtual screening uses a small molecule virtual database ZINC15 (http:// ZINC15.Docking. Org). The flexible docking program used in this experiment calculates the molecular energy state energy score between the small molecule ligand and the protein receptor, i.e. the van der waals energy and electrostatic energy between the small molecule and the receptor are evaluated by means of scoring. The experimental procedure was as follows: 1) Protein crystal structure: selecting a hSTING-CTD structure file 4KSY from a PDB database, deleting a ligand existing on a protein, removing metal ions and solvent molecules in the structure under a Dock Pre framework, and adding hydrogen atoms and charges on the structure to complete preparation of receptor protein molecules; 2) Preparation of ligand small molecules: after data containing a micromolecule structure are downloaded from the ZINC15, marvin software is used for hydrogenating the micromolecule structure, corina software is used for converting the micromolecule into a 3D space structure, and the energy state of the micromolecule monomer is calculated by AMSOL software; 3) Uploading the prepared ligand and protein crystal files to a computer server; 4) Generating Grid at the pocket where 2',3' -cGAMP binds to proteins for evaluation of energy status within this spatial region; 5) Calculating the grid energy state: AMBER is used to calculate van der waals energy and QNIFFT is used to calculate electrostatic energy; 6) Virtual screening: and carrying out molecular docking on the small molecular structures in the database in pockets of the receptor lattice points, checking docking results and judging the possibility of combining the protein and the compound.

10. Evaluation of cell viability

The toxic effects of the compounds in cells were tested using the Cell Proliferation Assay kit from Promega. The experimental procedure was as follows: 1) According to different growth speeds of the cells, different numbers of cells are spotted into a 96-well plate one day in advance; 2) Respectively incubating the compounds with different concentrations for corresponding time when the cells grow to the appropriate density of more than 75%; 3) After the compound incubation was completed and 20. Mu.l of MTS working solution was added to each well for reaction for 1 hour, absorbance was measured at a wavelength of 490nm and the effect of the compound on cell survival was calculated.

11. Transfection of cells

Two expression methods for transfecting target plasmids into cells are a calcium chloride-HEPES calcium transfer system and a liposome transfection system, wherein calcium transfer mainly aims at 293T cells, and stimulators such as ISD, HT-DNA and the like are mainly transferred into cytoplasm by liposome transfection. The calcium conversion method comprises the following steps: when the cells grow to about 70 percent of density, starting calcium transformation, and mixing the target plasmid with CaCl ₂ Mixing, adding HEPES solution, blowing, mixing, dropping into cell, and transfecting 2The plasmid was expressed intracellularly for 4 hours. The lipofection method is as follows: the stimulus was mixed with lipo2000 at 1:1, standing for 5 minutes, adding the stimulus into lipo2000 solution, mixing uniformly and adding into cells to complete transfection. After 6 hours of stimulation, the cells are harvested for subsequent experimental operations.

12. CDNs activate intracellular STING signaling pathways

Cells are grown adherently in culture plates, incubated overnight with the test compound at the corresponding concentration, and incubated with the medium containing 50mM HEPES (pH = 7.0), 100mM KCl,3mM MgCl ₂ 0.1mM DTT and 85mM sucrose buffer solution, dissolved CDNs stimulant, and (0.2% (m/v) BSA,1mM ATP,0.1mM GTP and 10 mu g/ml Digitonin) to prepare a working solution to incubate cell transmembrane so that CDNs molecules enter cytoplasm to activate STING protein on endoplasmic reticulum, and collecting the expression of cell detection related genes after the CDNs stimulate the cells for 3 hours.

13. Half maximal inhibition rate (IC) of test compounds in cells ₅₀ )

HFF cells were grown adherently in culture plates, incubation of compound was started when the cells grew to 80% abundance, and test compounds were incubated (compound dissolved in DMSO, initial final concentration of compound in culture medium: 1 and 10. Mu.M. IC, respectively ₅₀ And (3) testing concentration: 5,2.5,1.25,0.625,0.3125,0.15625,0.078125,0.039 μ M) overnight, a blank control with DMSO alone. After completion of incubation, cells were treated with digitonin solution containing 2'3' -cGAMP stimulator (2'3' -cGAMP final concentration: 1. Mu.g/mL) to allow 2'3' -cGAMP molecule to enter cytoplasm and further activate the STING protein on the endoplasmic reticulum, cells were harvested 3 hours after 2'3' -cGAMP stimulation, and expression of Ifnb gene was examined. The inhibition rate of the compound on the STING signal pathway after 2'3' -cGAMP stimulation at 1 and 10 μ M concentration was calculated according to the Ifnb gene expression fold: 1- [ Ifnb (Compound group)/Ifnb (DMSO group)]。IC ₅₀ Values were curve-fitted to the ratio of compound-treated versus DMSO blank to inhibit Ifnb gene expression.

14. Plasmid construction

The sequence information related to STING, TBK1, IRF3 and cGAS is inquired in Genbank of NCBI, and cDNA of plasmid is obtained from thymus cDNA library by using PCR method, and cloned into corresponding eukaryotic expression vector to obtain the corresponding plasmid. Point mutants of all plasmids used QuickChange XLSite-directed mutagenesis methods (Stratagene). The cloning method of the plasmid is as follows: 1) Designing a cloning primer corresponding to the plasmid fragment, and protecting a basic group-an enzyme digestion sequence-a target primer (20-25 bp); 2) Performing PCR amplification on a target gene sequence by using DNA polymerase KOD; 3) Recovering the amplified DNA product through agarose gel; 4) Carrying out double enzyme digestion and connection on the recovered product and the used carrier; 5) And (3) coating the ligation product, selecting positive clones, and extracting the correctness of the plasmid sequencing comparison to the target gene sequence to finish the cloning of the plasmid.

15. Immunoblotting method for detecting expression of target protein

The experimental procedure for the immunoblotting method (western blot) is as follows: 1) Cells were lysed with a protein lysate (0.5% TritonX-100,1mM EDTA,1% Cocktail dissolved in TBS buffer), then centrifuged at high speed, the supernatant lysate was taken to measure the protein concentration, and the protein concentration was measured using the BCA method protein concentration kit, and the volume and concentration of the sample were leveled based on the protein concentration; 2) Adding 5 x loading buffer into the sample to prepare a sample, and running SDS-PAGE gel with corresponding concentration according to the molecular weight of the target protein; 3) Transferring the membrane, and wet transferring the protein separated from the gel onto a PVDF membrane; 4) Blocking, normal proteins were blocked with 5% skim milk, phosphorylated proteins were blocked with 5% bsa; 5) First antibody incubation, after the antibody is diluted according to the corresponding concentration, the antibody is incubated at 4 ℃ for 2 hours or is overnight and then washed 6 times by TBST buffer solution, each time for 6 minutes, so as to wash off nonspecific adsorption; 6) Incubating the secondary antibody, diluting the secondary antibody with corresponding attribute according to corresponding concentration, incubating for 1 hour at room temperature, washing for 6 times by TBST buffer for 6 minutes each time, and washing out nonspecific adsorption; 7) And (4) developing, and detecting the expression of the protein by using a chemiluminescence strip of the protein by using ChemiDoc of Bio-Rad after ECL luminescent solution incubation.

16. Non-denaturing polyacrylamide gel electrophoresis analysis of protein dimerization and multimerization expression levels

The experimental method is as follows: 1) Preparing non-denatured polyacrylamide gel with corresponding concentration, wherein the gel does not contain SDS; 2) Pre-electrophoresing the prepared non-denatured glue in a buffer (25mM Tris,192mM Glycine, pH8.4, 0.2% sodium deoxycholate in internal trough) with 40mA for 30 minutes, and balancing the non-denatured glue with the electrophoretic solution; 3) After the cells were lysed with a protein lysate (0.5% TritonX-100,1mM EDTA,1% Cocktail dissolved in TBS buffer), 5 Xnative loading buffer was added to prepare samples; 4) Electrophoresis conditions are 25mA constant current for 1-1.5 hours, and subsequent detection is carried out according to a standard immunoblotting method.

17. Method for detecting interaction between proteins by co-immunoprecipitation

The interaction of proteins was detected by Co-immunoprecipitation (Co-IP) as follows: 1) Adding protein lysate (0.5% Triton X-100,1mM EDTA,1% Cocktail dissolved in TBS buffer) into cells, lysing, performing ultrasonic treatment until cell lysate is clarified, centrifuging at 13000rpm at 4 ℃ for 15 minutes, and collecting supernatant protein solution; 2) Specifically recognizing target protein, adding 1 mu l of target protein antibody into protein lysate, shaking up at 4 ℃ for 4 hours, and incubating; 3) Adding 20 μ l of prepared Protein G Agarose to the Protein lysate, returning to 4 deg.C, incubating for 2 hr to bind the Beads to the antibody, and washing off the non-specifically adsorbed Protein on Agarose with buffer (0.5% Triton X-100,1mM EDTA); 5) Preparing a sample, adding a 5 x loading buffer of the protein into the tube, denaturing the protein at 95 ℃ for 6 minutes, and detecting the expression of the protein of interest by a standard immunoblotting method to judge whether the target protein and the protein of interest have interaction.

18. Immunofluorescence

Immunofluorescence experiments on cells were performed as follows: 1) Plating cells on a cover glass to grow to a suitable density; 2) Incubating and stimulating the cells with the corresponding compounds, adding 4% paraformaldehyde, fixing at room temperature for 1 hr, washing with PBS twice, and penetrating the membrane with rupture buffer (0.25% Triton,1mM EDTA dissolved in PBS) for 20-30 min; 3) The slides were transferred to dark and blocked by addition of blocking solution (5% bsa solubilized in PBS) for at least 1 hour before incubating the primary antibody to the corresponding test protein (1; 4) After the primary antibody incubation is finished, adding PBS to wash off non-specific adsorption, adding a fluorescence labeled secondary antibody, and incubating for 1 hour at room temperature in a dark place; 5) Staining nuclei with DAPI for 1 min after incubation; 6) After nuclear staining, using an anti-fluorescence quencher for sealing, storing in dark place, and observing the fluorescence distribution of the protein in the cells under 63x NA1.4 oil lens of a laser confocal microscope Carl Zeiss LSM 700.

19. Prokaryotic expression and purification of His-hSTING-CTD (149-379) protein

The experimental method for expressing the target protein fragment in Escherichia coli is as follows: 1) Constructing an expression vector plasmid His-STING-149-379 containing a target protein fragment; 2) Transforming 1 mu g of plasmid, transferring, and inducing the protein to be expressed in a large amount in escherichia coli by IPTG with the final concentration of 0.5mM at 18 ℃; 3) After bacteria are collected by using buffer solution (25mM Tris8.0, 150mM NaCl, pH = 8.0), the bacteria are crushed by ultrasonic for 15 minutes until the bacteria solution is slightly clear; 4) Centrifuging at high speed (18000rpm, 30 min), and collecting upper layer protein solution; 5) Hanging 1.5ml of Ni column to adsorb the target protein on the Ni column, and then washing off the non-specific binding; 6) Eluting the target protein from the Ni-NTA Agarose by gradient imidazole, 80mM,250mM and 500mM; 7) Running SDS-PAGE gel, staining with Coomassie brilliant blue, and detecting the expression of the target protein; 8) Collecting a target protein solution, concentrating the protein solution by using a 50KD ultrafiltration tube, passing the concentrated protein solution through an Akata protein purification system, removing foreign proteins and collecting the target protein; 9) His-STING-149-379 protein for crystallization experiment is subjected to His-tag cleavage by TEV enzyme in advance, and then is subjected to molecular exclusion chromatographic column purification to obtain target protein without tag, and then is concentrated to the concentration of 15 mg/ml; 10 ) concentrating the purified target protein to a proper concentration, subpackaging, quick-freezing with liquid nitrogen, and storing at-80 deg.C.

20. Co-crystallization of SN-011 and hSTING-CTD (149-379) protein, data processing and structure analysis

15mg/ml of STING-CTD (149-379) protein was dissolved in 25mM Tris-HCl, pH 7.5,150mM NaCl, and 1. Mu.L of SN-011 (50 mM) was added to 100. Mu.L of the protein solution, and after incubation for 1 hour, the supernatant protein solution was centrifuged at high speed and used for crystallization. mu.L of the protein solution was mixed with 1. Mu.L of a pool solution (0.1M HEPS-NaOH, pH 7.0,0.1M sodium form, 25% PEG3350) using the pendant drop method, crystals were grown in a 16 ℃ environment, and the crystals were harvested and stored in liquid nitrogen using a cryopreservation solution containing 30% PEG 3350. The X-ray diffraction is carried out in the Shanghai national center for light sources, and the acquisition, integration and processing of diffraction data are carried out by using HKL3000 software and using apo-STING (PDB: 4F 5W) as a template for carrying out structural analysis by using a molecular replacement method. The co-crystal structure was adjusted using COOT software and the structure was refined using CCP4 software.

21. Surface Plasmon Resonance (SPR) technology for detecting affinity between SN-011 and His-STING-CTD (149-379) protein

The SPR experimental temperature is 25 ℃, and the experimental instrument is Biacore T200 SPR instrument (GEHealthcare). Purified hSITNG-CTD protein at 2. Mu.g/ml and its mutant were immobilized on a Sensor Chip CM5 (carboxmethylated dextran surface) Chip using Amine Coupling Kit under ph = 5.5. SN-01 gradient dilution (nM): 15.6,7.81,3.91,1.85,0.93 and cGAMP gradient dilution (nM): 125,62.5,31.3,15.6,7.81 at 30. Mu.l min ^-1 The speed of (2) was 90s for all the binding dissociation time. The buffers used in the experiments were all PBS containing 0.05% Tween 20. All data were repeated twice under the same conditions. The final affinity data was fitted using the 1:1 binding model in the software Biacore T200 evolution software version 3.0 (GE Healthcare).

22. Rotating rod test (Rotarod test)

The test was carried out using an XR1514 model rat and mouse rotary rod fatigue tester (purchased from Shanghai Xin soft information technology Co., ltd.), the set rotation speed was 24r/min, the maximum recording time was 5min, and 5min was uniformly registered for over 5min. Mice were trained 3 times prior to the formal experiment. The formal experiment is to record the movement time of the mouse on the rotating rod, and the average value is taken as the final result after 3 times of tests.

23. Morris Water maze test

1) And putting the mouse head towards the wall of the pool into water, putting the platform in the fourth quadrant, and putting the mouse head towards the wall of the pool into the pool from any point of the four starting points of the wall of the pool. The time(s) when the animal found the underwater platform was recorded. In the first few training sessions, if this time exceeded 60s, the mice were directed to the platform. Let it stay on the platform for 10s.

2) The mice were removed and wiped dry. Each mouse is trained in water from different quadrants every day, the interval between two times of training is 15-20 min, and the training is continuously carried out for 5 days.

3) The next day after the last acquisition training, the platform was removed and the exploration training started for 60 s. Mice were placed in the water from the opposite side of the original platform quadrant. Recording the time spent by the mouse in a target quadrant (the quadrant where the platform is originally placed) and the proportion of the total time and the movement distance of the mouse in the target quadrant, and taking the time and the proportion as a detection index of space memory;

24. RNA extraction and real-time fluorescent quantitative PCR technology for detecting expression of target gene

The process of extracting total RNA from cells or tissues is as follows: 1) After taking a proper amount of cells and tissues and fully cracking by TRIzol (Invitrogen), 1:5 is added into chloroform to extract RNA in a lysate, and the lysate is centrifuged at 12000g for 15 minutes at 4 ℃; 2) Adding isopropanol with the same volume into the upper aqueous phase solution to precipitate the RNA in the solution; 3) Washing the RNA precipitate with 1ml of 75% ethanol to remove impurities; 4) After drying and clearing, the RNA was dissolved in an appropriate amount of DEPC water at 55 ℃ and the RNA concentration was measured at OD 260. The total RNA extraction can be used for the subsequent fluorescent quantitative PCR detection of the expression of the target gene after the total RNA extraction is completed, and the experimental steps are as follows: RNA and primer Oligo dT are mixed and then transcribed into cDNA under the action of a reverse transcriptase kit, the cDNA and a primer of a target gene, DNA polymerase and fluorescent dye FastStart Universal SYBR GREEN MASTER MIX (Roche) are mixed and then amplified and detected in an ABI Quantstudio 3 instrument, GAPDH is an internal reference of gene expression, 2 ^-ΔΔCT The method carries out relative quantitative calculation of the expression of the target gene. The sequences of primers used to detect the gene of interest are shown in table 1 below:

TABLE 1 qPCR primer sequences

25、RNA-Sequencing

WT or Trex1 from 6 to 8 weeks ^-/- After isolating bone marrow cells from the femoral femur of the mouse, the cells were induced to differentiate into BMDMs using conditioned medium containing L929 supernatant. After induction was complete, SN-011 (500 nM) was incubated for 12 hours, respectively, according to the experimental group design, RNA was extracted from the cells and integrated into the cDNA library according to the standard Illumina RNA-seqperocol. The resulting cDNA library was sequenced with Illumina HiSeq2000 at 1X 100 bp run, processed with the sequencing results and compared to the mouse genome for differences in expression of the thermographic response genes and further cluster analysis.

Example 1

N- (3- ((4-fluorophenyl) sulfonylamino) -4-hydroxyphenyl) - [1,1' -biphenyl ] -4-carboxamide (Compound I)

Compound 1 (2.00g, 13.0 mmol) and pyridine (1.54g, 19.5mmol, 1.57mL) were dissolved in 40mL of dichloromethane, 4-fluorobenzenesulfonyl chloride 1A (3.04g, 15.68mmol) dissolved in 20mL of dichloromethane was added dropwise at 0 ℃, mixed, and the reaction was stirred at 25 ℃ for 12 hours. The reaction was then identified as complete by Thin Layer Chromatography (TLC) with the mobile phase being petroleum ether, ethyl acetate =2:1. The solvent was evaporated under reduced pressure and the crude product was purified by silica gel chromatography (mobile phase was petroleum ether: ethyl acetate =10: 1-2. ¹ H NMR:(DMSO-d ₆ ,400MHz)δ8.04(d,J＝2.4Hz,1H),7.94～7.92(m,1H),7.83～7.80(m,2H),7.41～7.37(m,2H),6.89(d,J＝8.8Hz,1H).LCMS(m/z):312.02[M+H] ⁺ .

Dissolving Compound 2 (1.50g, 4.80mmol) in 15mL of methanol, adding 10% Pd/C (0.1 g), reacting for 16 hours at 25 ℃ in a hydrogen atmosphere, and identifying the reaction product by LC-MS to judge the completion of the reaction. The reaction solution was filtered through a celite packed column, and the solvent was removed under reduced pressure to obtain 1.4g of a yellow solid product, compound 3. ¹ H NMR:(DMSO-d ₆ ,400MHz)δ8.44(br.s,1H),7.81～7.77(m,2H),7.37～7.33(m,2H),6.51(d,J＝2.4Hz,1H),6.42(d,J＝8.4Hz,1H),6.19(dd,J1＝2.4Hz,J2＝8.4Hz,1H),4.60(br.s,1H),3.17(s,1H).LCMS(m/z):282.05[M+H] ⁺ .

990mg of Compound 3 was dissolved in 10mL of methylene chloride, followed by addition of 229mg of imidazole and dropwise addition of 507mg of TBSCl (t-butyldimethylsilyl chloride) dissolved in 1mL of methylene chloride. The reaction is stirred for 12 hours at 15 ℃, and the LC-MS identifies the reaction product to judge that the reaction is complete. The reaction mixture was washed with water (20mL. Times.3), anhydrous Na ₂ SO ₄ Drying and evaporating the solvent under reduced pressure. Al for crude product ₂ O ₃ And (3) performing packed chromatography (a mobile phase is dichloromethane: methanol =10:1,v/V) to obtain a reddish brown solid compound 4. ¹ H NMR:(CDCl ₃ ,400MHz)δ7.78～7.75(m,2H),7.10～7.06(m,2H),6.97(d,J＝2.8Hz,1H),6.78(s,1H),6.52(d,J＝8.4Hz,1H),6.28(dd,J1＝2.8Hz,J2＝8.8Hz,1H),3.48(s,2H),0.93(s,9H),0.08(s,6H).LCMS(m/z):396.13[M+H] ⁺ .

To 5mL of methylene chloride was added 185mg of Compound 4A,215mg of EDCl (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), 139mg of HOBt (1-hydroxybenzotriazole) and 181mg of DIPEA (N, N-diisopropylethylamine), and finally 370mg of Compound 4. The reaction is stirred for 16 hours at the temperature of 15 ℃, and the LC-MS identifies the reaction product to judge that the reaction is complete. The reaction product was diluted with 10mL of dichloromethane, washed with water (20mL. Times.3), anhydrous Na ₂ SO ₄ Drying and removal of the solvent by evaporation under reduced pressure gave 400mg of the crude compound 5 as a reddish brown solid which was used in the subsequent reaction without further purification.

To 15mL of dichloromethane were added 630mg of Compound 5 and 176mg of Et ₃ And N.3HF, stirring the reaction at 15 ℃ for 3 hours, and identifying the reaction product by LC-MS to judge that the reaction is complete. The reaction mixture was diluted with 25mL of dichloromethane, washed with water (20mL. Times.2), and washed with anhydrous Na ₂ SO ₄ Drying and evaporating the solvent under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase petroleum ether: ethyl acetate = 5:1) to give compound I as an off-white solid (260mg, 51.5% yield). ¹ H NMR:(DMSO-d ₆ ,400MHz)δ10.13(s,1H),9.38(s,2H),8.03(d,J＝8.4Hz,2H),7.83～7.71(m,7H),7.51～7.35(m,6H),6.70(d,J＝8.8Hz,1H).LCMS(m/z):462.10[M+H] ⁺ .

Example 2

N- (3- ((4-fluorophenyl) sulfonylamino) -4-methoxyphenyl) - [1,1' -biphenyl ] -4-carboxamide (Compound II)

Compound 6 (0.54g, 3.2mmol), pyridine (0.5 mL) was added to dichloromethane (10 mL), and a solution of 4-fluorobenzenesulfonyl chloride (1A. The solvent was evaporated under reduced pressure, ethyl acetate (50 mL) and water (20 mL) were added, the layers were shaken well, and the organic phase was washed with 1N HCl (10 mL), water (20 mL) and saturated brine (20 mL), anhydrous Na ₂ SO ₄ Drying, filtering, and distilling off the solvent under reduced pressure to obtain a residue, and purifying the crude product by using a silica gel chromatographic column (the mobile phase is petroleum ether: ethyl acetate =10 = 1-2.

Compound 7 (0.5g, 1.53mmol), 10% Pd/C (50 mg) was added to MeOH (20 mL), H ₂ And reacting for 12 hours in an atmosphere. Pd/C was filtered off, the filter cake was washed with MeOH, and the solvent was distilled off from the filtrate under reduced pressure to give compound 8 (0.43 g) as a tan solid product.

Compound 4A (119mg, 0.60mmol) and DIPEA (0.16mL, 0.96mmol) were added to THF (4 mL), HATU (O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) (291mg, 0.77mmol) was added and reacted at room temperature for 1h, followed by addition of compound 8 (237mg, 0.80mmol) and reaction at room temperature for 5h. Ethyl acetate (20 mL) and water (10 mL) were added, the layers were separated, and the organic layer was washed with 1N HCl (5 mL), water (10 mL) and saturated brine (10 mL), anhydrous Na ₂ SO ₄ Drying, filtering, evaporating under reduced pressure to remove solvent to obtain residue, and purifying by silica gel column chromatography (mobile phase is petroleum ether: ethyl acetate = 5:1) to obtain off-white solid compound II (117mg, 41%). ¹ H NMR:(DMSO-d ₆ ,400MHz)δ10.25(s,1H),9.59(s,1H),8.15(d,J＝8.4Hz,2H),7.87～7.73(m,7H),7.65～7.35(m,6H),6.88(d,J＝8.8Hz,1H)，3.48(s,3H).LCMS(m/z):477.5[M+H] ⁺ .

Example 3

4- ([ 1,1' -biphenyl ] -4-carboxamido) -2- ((4-fluorophenyl) sulfonamido) phenylacetate (Compound V)

Compound I (200mg, 0.43mmol) prepared from example 1 was dissolved in THF (5 mL), pyridine (52. Mu.L, 0.65 mmol) was added, acetyl chloride (37. Mu.L, 0.52 mmol) was added dropwise at room temperature, and the reaction was carried out at room temperature for 10h. Quenched with water (10 mL), extracted with EtOAc (30 mL), and the organic phase washed sequentially with 1N HCl (10 mL), water (10 mL), and saturated brine (10 mL) over anhydrous Na ₂ SO ₄ Drying, filtration and evaporation of the solvent under reduced pressure gave a white solid which was purified by silica gel column chromatography (DCM/MeOH = 10/1) to give compound V (76mg, 35%) as a white solid. ¹ H NMR(300MHz,DMSO-d ₆ )δppm 10.39(s,1H),10.01(s,1H),8.06(d,J＝8.3Hz,2H),7.96(d,J＝2.2Hz,1H),7.86-7.8(m,4H),7.77(d,J＝7.4Hz,2H),7.62(dd,J＝8.8,2.3Hz,1H),7.52(dd,J＝7.4,7.4Hz,2H),7.44(d,J＝5.3Hz,1H),7.40(dd,J＝7.9,7.9Hz,2H),7.01(d,J＝8.8Hz,1H),2.14(s,3H).ESI-MS:m/z 461.1[M-CH ₃ CO] ^- .

Example 4

4'- ((3- ((4-fluorophenyl) sulfonamido) -4-methoxyphenyl) carbamoyl carboxamido) - [1,1' -biphenyl ] -4-yl acetate (Compound III)

Compound III was prepared according to the procedure of example 2: ESI-MS [ m/z ] 491.3[ m-CH ] ₃ CO] ^- .

Example 5

N- (4-methoxy-3- (phenylsulfonamido) phenyl) - [1,1' -biphenyl ] -4-carboxamide (Compound IV)

Compound IV was prepared according to the procedure of example 2: ESI-MS: m/z 459.1[ 2 ] M + H] ⁺ .

Example 6

Virtual screening and in vitro activity evaluation based on STING protein crystal structure

The invention carries out computer simulation screening of potential small molecules combined with STING based on the analysis of the crystal structure of the C-terminal domain of the human STING protein (hSTING-CTD-139-379, PDB. And (3) performing butt screening on the small molecules in a ZINC15 (http:// ZINC15.Docking. Org) virtual compound database and the hSTING-CTD structure by using DOCK3.7 software, wherein 152 small molecules are used for responding to the combination of the hSTING-CTD after the screening is completed. The small molecular compounds screened by analysis discover that the small molecular compounds containing 1-sulfanilamide-3-amide-benzene ring skeleton structure have good responsiveness to the hSTING-CTD structure. On the basis, the micromolecular compound with the characteristic structure is purchased from EnamineStore company for in vitro biological activity evaluation. As a result, it was found that the compound of the formula I (SN-011), the compound of the formula II (SN-001), the compound of the formula III (SN-005), the compound of the formula IV (SN-006) and the compound of the formula V (SN-010) can significantly inhibit the expression of the immunoinflammatory factor gene such as Ifnb (FIG. 1A, FIGS. 2A to 2H, FIG. 3A, FIG. 3C and FIG. 3E) in MEF cells or L929 cells in which stimulation is induced by DNA analogs such as ISD or HT-DNA, etc., and the expression of the Ifnb gene in a resting state (FIG. 1B) is not affected. The compound SN-100 (CAS: 1384744-19-7) did not affect ISD-induced expression of IFN- β genes in MEF cells, and therefore, this compound was selected as a negative control compound for subsequent experiments. Furthermore, in HT-DNA stimulated L929 cells SN-001 significantly inhibited STING, phosphorylation expression of TBK1, IRF3, P65, I κ B α and dimerization of IRF3 protein (FIG. 2I), and subsequently nuclear entry of transcription factors IRF3 and P65 was also significantly reduced after incubation of the compounds (FIGS. 2J-2K), whereas the negative control compound SN-100 did not affect phosphorylation expression of the above proteins and nuclear entry of transcription factors. To exclude the effect of SN-001 cytotoxicity on its biological activity, the effect of compounds on cell viability was examined by MTS assay in MEF, L929 and THP-1 cells, and the results showed that SN-001 (5. Mu.M-20. Mu.M) did not affect cell viability until 24 hours of incubation (FIGS. 2L-2N). Fig. 1C and 3B show the structures of a part of compounds purchased after virtual screening. It should be noted that the compounds such as the compound ZINC72311784 in fig. 1A and 1B are all commercially available compounds (some of the structures of the compounds are not shown). The CAS numbers for some of the compounds in figures 1 and 3 are as follows: SN-001/ZINC08686914 (CAS: 727699-84-5), SN-003/ZINC15418850 (CAS: 1385921-05-0), SN-004/ZINC00991157 (CAS: 681834-84-4), SN-007 (CAS: 568569-75-5), SN-008 (CAS: 2249106-01-0), SN-100/ZINC78992473 (CAS: 1384744-19-7).

In MEF cells, pre-incubation compound SN-011 (compound of formula I) significantly down-regulated the expression of Ifnb, cxcl10 and Il-6 genes induced by the above stimuli using the classical agonists of the STING signaling pathway, ISD, HT-DNA, HSV-1,c-di-GMP and 2'3' -cGAMP (FIGS. 3C-3E).

Further by evaluating the half inhibition rate (IC) of SN-011 (compound of formula I) in different cells ₅₀ ) The in vitro biological activity of the compounds was studied. SN-011 IC in MEF (mouse embryonic fibroblasts), BMDM (mouse bone marrow primary macrophages) and HFF (human primary foreskin fibroblasts) ₅₀ The values were 127.5. + -. 5.5nM, 121.7. + -. 32.2nM and 502.8. + -. 94.5nM, respectively (FIGS. 4A-4C). SN-011 also has excellent safety in vitro, as demonstrated by no effect on cell viability of compounds incubated in MEF, BMDM and HFF cells up to 20 μ M for 48 hours (FIGS. 4D-4F). Pre-incubation of SN-011 in STING knockout MEF cells did not affect the expression of LPS, cpG-DNA, poly (I: C) and Sendai Virus activated IFR3 and NF-. Kappa.B downstream genes Ifnb, tnf-alpha and Il-6 (FIGS. 5A-5B). In addition, preincubation of SN-011 did not affect Ifn β activation of expression of Isg15 and Isg56 genes in STING knockout MEF cells (fig. 5B).

The results show that the compounds shown in the formulas I to V can efficiently, safely and specifically inhibit the activation of a cGAS-STING signal pathway.

Example 7

SN-011 (Compound of formula I) inhibits the activation of STING protein and its mediated downstream signaling

In a resting state, the STING protein is anchored on an endoplasmic reticulum in a homodimer form through an N-terminal transmembrane region (amino acid 1-137), and after STING is combined with a CDNs molecule serving as a ligand thereof, such as 2',3' -cGAMP, the spatial conformation of the STING dimer is changed, specifically, after the C-terminal structure (amino acid 138-379) rotates 180 degrees relative to the transmembrane region, a plurality of dimer molecules form a tetramer and a higher multimer form in a side-by-side arrangement mode (Nature, 2019,567 (7748): 389-393). The multimeric STING proteins migrate from the endoplasmic reticulum to the golgi apparatus, during which the STING multimer recruits the kinase protein TBK1 with the plplplrt/SD sequence in its C-terminal region and promotes autophosphorylation activation of TBK1 (Ser 172) (Nature, 2019,567 (7748): 394-398, nature,2019,569 (7758): 718-722. Activated TBK1 further phosphorylates serine in the pLxIS366 sequence of the STING multimer, thereby facilitating the recruitment of the transcription factor IRF3, and IRF3 is further phosphorylated and activated by TBK1 after being recruited to the multimeric complex (Science, 2015,347 (6227): aaa2630; proceedings of the National Academy of Sciences,2016,113 (24): E3403-E3412), which is the activation of protein levels of STING and the process of mediating downstream signaling. Based on this, it was found that pre-incubation of SN-011 significantly inhibited 2',3' -cGAMP-induced polymerization, phosphorylation of STING in HFF cells (FIG. 6A) and its transfer to Golgi (FIG. 6E). In addition, SN-011 was able to significantly reduce recruitment of STING proteins to downstream TBK1 and IRF3 (fig. 6B), consistent with 2',3' -cGAMP-induced phosphorylation of TBK1, IRF3, P65 and I κ B α proteins and dimerization levels of IRF3 (fig. 6C) and nuclear import of IRF3 significantly decreased after pre-incubation of SN-011 in HFF cells (fig. 6D). In contrast, the negative control compound SN-100 did not affect the changes in the protein levels described above. As a control, the influence of SN-011 on the stability of cGAS, STING, TBK1 and IRF3 proteins in the resting state of cells was further examined, and the results showed that SN-011 does not affect the ground-state expression of the above proteins in HFF cells (FIG. 5C), and the expression of cGAS gene (MB 21D 1) and STING gene (TMEM 173) (FIGS. 5D to 5E). This suggests that the mechanism of action of SN-011 is by directly affecting the function of the protein rather than affecting the stability of the STING signaling pathway protein. The above studies indicate that SN-011 can inhibit the activation of intracellular STING protein and its mediated downstream signaling.

Example 8

Co-crystal structure of SN-011 (compound of formula I) and STING-CTD (149-379) protein

In order to study the structural basis of the binding of SN-011 and STING-CTD (149-379) protein, the invention obtains and analyzes the cocrystal structure of SN-011 and STING-CTD (149-379) protein with the resolution

The invisibility of the C-terminal region (amino acid 341-379) in the cocrystallized structure may be due to the spatial instability of the structure. Each corresponding building block in the cocrystal structure contains a homodimer of two STING protein monomers and two SN-011 molecules bound in a protein dimer CDNs binding pocket (fig. 7A). Structurally, SN-011 molecules bind in anti-phase and parallel to the surface of STING dimer, biphenyl rings on small molecule structures bind at the bottom of dimer pocket, and 4-fluoro-benzenesulfonamide groups extend to the top of protein dimer pocket. Similar to the crystal structure of apo-STING-CTD protein, STING-CTD (149-379) maintains a V-shaped dimer structure after binding to SN-011, and has His185 amino acids at a distance from the side of the pocket

(FIG. 7B). In contrast, after the STING-CTD protein is combined with 2'3' -cGAMP and c-di-AMP, the monomers on both sides of the pocket approach each other under the traction of small molecules, so that the protein dimer pocket becomes a closed 'U' shape and the distance between His185 amino acids on both sides of the pocket is shortened to be

(FIG. 7C) (Cell, 2019, 178. Subsequently, the protein STING-CTD after SN-011 binding was further analyzedThe co-crystal structure is packed in the lattice in a manner similar to "head-to-tail" packing (FIG. 7D), which is also present in the inactivated STING structure (ACS Med Chem Lett,2019,10 (1): 92-97), and which is significantly different from the "side-by-side" packing of STING-CTD proteins in the lattice after 2'3' -cGAMP and c-di-AMP are bound (Nature, 2019,567 (7748): 389-393). In conclusion, after binding to the SN-011 molecule, the steric conformation of STING protein is not significantly changed, and still maintains its dimeric configuration in the resting state.

Further structural analysis indicates that SN-011 is stabilized within the pocket by forming hydrogen bonds and stacking with specific amino acids within the pocket of the STING protein dimer. The biphenyl ring in the structure of SN-011 and the benzene ring of the amino acid side chain of protein Tyr167 form pi-pi stacking effect. The benzene ring connected with the phenolic hydroxyl in the SN-011 structure can also form conjugation or hydrogen bond interaction with the surrounding Glu260 and Ser241 amino acids for stabilization. While the phenolic hydroxyl and the sulfonamide bond in SN-011 can further form hydrogen bond with the hydroxyl on the Ser243 amino acid side chain (FIG. 7E). Therefore, the co-crystal structure of the SN-011 and the STING protein analyzed by the invention not only defines the binding position of the compound in the protein, but also provides the interaction mode and binding site information of the two.

The above results indicate that the compounds of the present invention are capable of inhibiting the activation of STING signaling pathway by directly binding to STING protein and maintaining its dimeric conformation in resting state.

Example 9

Mutating the amino acid site that binds to SN-011 (a compound of formula I) affects the binding of small molecules and the biological activity exerted by them

To further determine the effect of amino acid sites on SN-011 binding as analyzed by cocrystallization, the effect of mutations at the relevant amino acid sites on the affinity between small molecules and proteins was examined by Surface Plasmon Resonance (SPR). The detection result of SPR shows that the affinity between SN-011 and hSTING-CTD protein is about 4.03nM, and is better than the affinity (Kd-9.23 nM) for 2'3' -cGAMP to bind. Notably, the single mutant S243A and the triple mutant proteins S241A, S243A and T263A of the hSTING-CTD binding site significantly attenuated the interaction between hSTING-CTD and SN-011 with Kd of 176.2nM and 1.36 μ M, respectively (fig. 8A-8E). hSTING-CTD-Y167A and hSTING-CTD-E260A cannot be purified in Escherichia coli due to solubility problems, so the influence of Y167 and E260 on the affinity between small molecules and proteins cannot be evaluated in this experimental system.

Subsequently, the hSTING protein and its related point mutant proteins were overexpressed in HEK293T cells to evaluate the effect on the biological activity of SN-011 after binding site mutation. Experimental results show that compared with wild hSTING, the inhibitory activity of SN-011 on IFN-beta gene expression induced by point mutants S241A, S243A, T263A and 3A is remarkably reduced, and the inhibitory activity of SN-011 can be weakened by mutating the amino acid sites under the stimulation condition of 2'3' -cGAMP (FIGS. 8F-8I). The hSTING-Y167A and hSTING-E260A mutants can not activate the downstream IFN-beta gene expression after being over-expressed in HEK293T cells, and can not respond to the stimulation of exogenous 2'3' -cGAMP (8G, 8I), so the influence of the mutations at the two sites on the biological activity of SN-011 can not be evaluated. The research results show that the compound disclosed by the invention is combined with the STING protein through a plurality of key amino acid residues (Tyr 167, glu260, ser241 and Ser 243) so as to inhibit the activation of the STING signal pathway. Therefore, the benzene sulfonamide compounds shown in the formulas I to V or the pharmaceutically acceptable salts or solvates thereof can be used for preparing the medicines for inhibiting the activation of the STING signal pathway.

Example 10

SN-011 (Compound of formula I) improves TREX1 ^-/- Systemic inflammatory injury in autoimmune disease mice

TREX1 plays a role of exonuclease in cytoplasm and is used for degrading abnormal ssDNA in cytoplasm, and after gene mutation of the abnormal ssDNA causes protein to lose the function of normal enzyme, DNA accumulated in the cytoplasm can activate a cytoplasmic cGAS-STING signal pathway, so that the expression of I-type IFNs is induced, and the occurrence of cell inflammation is promoted. It was found in animal models that knocking out the expression of mouse cGAS or STING proteins significantly improved the extent of inflammatory lesions and the fatality of disease induced by TREX1 mutations in various tissues and organs (Nat Genet,2007,39 (9): 1065-7 cell,2008,134 (4):587-98). Based on this, the invention is in Trex1 ^-/- The potential pharmacodynamic activity of SN-011 on systemic inflammatory injury was evaluated in vivo in mice. First, in Trex1 ^-/- After the compound SN-011 is incubated in macrophages (BMDMs) derived from mouse bone marrow, the RNA-Seq technology is used for detecting and analyzing mRNA transcripts in cells, and the experimental result shows that: (1) In Wild Type (WT) mouse BMDMs, the expression of ISGs genes in a cell resting state cannot be significantly influenced by incubating SN-011; (2) Trex1 compared to WTBBMDMs cells ^-/- The expression level of ISGs in BMDMs cells is obviously improved, and the expression of related ISGs genes can be obviously inhibited after SN-011 incubation; (3) Further analysis of the changes in gene expression of cellular transcripts revealed that SN-011 also predominantly affects ISGs gene expression in cells, flanking the specificity of action of this compound (FIG. 9A). Subsequently, the expression conditions of Ifnb, cxcl10, isg15 and IL-6 genes in cells are detected by using a qPCR technology, and the detection result further verifies that the compound SN-011 has Trex1 ^-/- Inhibition of ISGs gene expression in BMDMs cells (FIGS. 9B-9E).

On the basis of in vitro studies, SN-011 (5 mg/kg) was injected intraperitoneally into Trex1 ^-/- In mice, inflammatory infiltrates of heart, stomach, tongue and muscle were detected after 1 month of continuous dosing. The results show that: (1) Trex1 compared to Wild Type (WT) mice ^-/- The mouse has remarkable increase of the immune (Ifn beta, cxcl10, isg 15) and inflammatory factor (Il 6) genes of the heart, the stomach, the tongue and the muscle; (2) Administration of compound SN-011 in wild-type mice did not affect the expression of immune and inflammatory factor genes in the above tissues; (3) Compared with Trex1 ^-/- The autoimmune disease model group of (1), administration of compound SN-011 can significantly reduce Trex1 ^-/- Immune (Ifn β, cxcl10, isg 15) and inflammatory factor (Il 6) gene expression in heart, stomach, tongue and muscle of mice (fig. 10A-10D). Further carrying out pathological section evaluation on the tissue and organ to evaluate the tissue inflammatory injury, and the result shows that SN-011 can obviously improve Trex1 ^-/- Inflammatory injury and immune cell infiltration of mouse tissues (fig. 10E). Spleen cells of experimental mice are taken and dispersed, T cell antibody staining is carried out, and the content of T cell subtype is detected by a flow cytometer. Detection ofThe test result shows that: trex1 compared to control ^-/- Mice, i.p. injected with compound SN-011, significantly reduced activated CD 8T (CD 69) ⁺ ) Cellular and memory CD 4T (CD 62L) ^low CD44 ^high ) Cells and CD8 (CD 62L) ^low CD44 ^high ) T cells in Trex1 ^-/- Mouse spleen content without affecting activated CD 4T (CD 69) ⁺ ) Cell content (FIGS. 11A-11B). In addition, SN-011 Trex1 post-administration ^-/- Antinuclear antibodies in mouse serum were significantly reduced (fig. 11D), and continued administration for 1 month significantly extended Trex1 ^-/- Survival of mice (fig. 11C). The research results show that SN-011 can improve TREX1 ^-/- Autoimmune disease mice systemic inflammatory injury and arrest disease progression. Further, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006), and the compound of formula V (SN-010) all have effects similar to those of the compound of formula I (SN-011).

In conclusion, the compounds of the formulae I to V of the present invention can be used for preparing drugs for preventing or treating STING-mediated autoimmune diseases and inflammatory diseases.

Example 11

Pharmacokinetic study of SN-011 (Compound of formula I) in mice

After a single intraperitoneal injection of compound SN-011 (5 mg/kg) in male C57BL6J mice, blood was collected at different time points within 24 hours, and then plasma concentrations were measured by LC-MS/MS and related pharmacokinetic parameters were calculated. The experimental results show that: half-life period T of drug in plasma after mice are injected with SN-011 through intraperitoneal injection _1/2 Is 1.03 hours, the maximum blood concentration Cmax is 837.02ng/mL, and the time T required for reaching the drug peak concentration _max The plasma concentration-time curve area AUC was 850ng/mL at 0.25 hr (FIGS. 12A to 12B).

Example 12

Toxicological evaluation of SN-011 (Compound of formula I) in mice

In order to evaluate the potential toxicity of the compound SN-011 after long-term administration in mice, SN-011 (1 mg/kg) is intraperitoneally injected into male C57BL6J mice for three times per week and is continuously administered for 10 weeks, and changes of organs and serum biochemical indexes in the mice are detected after the administration is completed. The experimental results show that: there was no significant change in body weight in mice from the control and the administered groups within 10 weeks of administration (fig. 13A), nor was there a significant increase in ALT and AST indicators in the mouse serum in response to liver injury after administration (fig. 13B). In addition, serum indicators of BUN and CREA in response to renal injury also did not change significantly after dosing (fig. 13C). Pathological sections of the mouse heart, kidney, stomach and liver showed no significant substantial damage to any of these organs after 10 weeks of dosing (fig. 13D). The results show that the compound SN-011 has good safety in mice, and does not show toxic or side effect in mice after long-term administration.

Example 13

SN-011 (Compound of formula I) inhibits the expression of inflammatory cytokines induced by SAVI-related STING point mutations

The gene TMEM173 encoding the STING protein is mutated in humans to induce a class of autoimmune diseases, i.e., STING-activated vasculopathy (STING-associated vasculopathy with on-set in infancement, SAVI) in infancy. Clinical manifestations of SAVI patients are mainly early infancy, systemic inflammation such as skin rash and tachypnea, fever, peripheral vascular lesions, lung inflammation and autoimmune antibodies in blood. Subsequent gene sequencing revealed that the TMEM173 gene of such patients was mutated at multiple sites to cause protein self-activation, including N154S, V155M, G166E, C206Y, R281Q and R284G (J Allergy Clin Immunol,2017,140 (2): 543-552 and Ann Rheum Dis,2017,76 (2): 468-472). The invention constructs the plasmid of the point mutant by a cloning method, over-expresses the mutant in HEK293T cells (without the expression of STING protein), and further researches the influence of the compound SN-011 on the activation of STING signal channels induced by the mutant. Experimental results show that the SN-011 can obviously inhibit the Ifnb, cxcl10 and Tnfa gene expressions induced by the mutant (figure 14A-14C). Pre-incubation of SN-011 at the protein level also attenuated the recruitment of overexpressed STING point mutants to downstream target proteins TBK1 and IRF3 and reduced the expression levels of P-TBK1 and P-IRF3 (FIGS. 14D-14E). Recent studies have shown that SAVI-related STING point mutants can activate protein multimerization independently of ligand binding by releasing the self-inhibitory form of STING dimers (Nature, 2019,567 (7748): 389-393), and based on this, the influence of SN-011 on multimerization of the point mutants was further examined, and as a result, SN-011 was shown to significantly inhibit expression of multimerization of STING mutants (FIG. 14F). In summary, SN-011 can reduce the expression of downstream inflammatory cytokines by inhibiting the polymerization activation of SAVI-associated STING point mutations, thereby reducing inflammatory injury to tissues or cells. Further, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006), and the compound of formula V (SN-010) all have similar effects to the compound of formula I (SN-011).

Therefore, the compounds of formula I-V of the present invention can be used for preparing a medicament for preventing or treating STING-related vascular disease (SAVI) occurring in infancy.

Example 14

SN-011 (compound of formula I) improves cerebral ischemia injury of rats

Ischemic injury of the tissue is accompanied by death of a large number of parenchymal cells, with the consequent release of injury-associated molecular patterns (DMAP) that further promote activation of the immune system at the site of injury, with the release of inflammatory factors that exacerbate tissue injury. Recent studies have found that cell debris generated by damaged cardiomyocytes in a mouse myocardial ischemia model is taken up by macrophages in heart tissues, an intrinsic immune signal pathway mediated by cGAS-STING in the macrophages is activated, the expression of ISGs is promoted, and the cGAS or STING is knocked out, so that heart inflammation damage caused by myocardial ischemia can be obviously improved, the heart function is improved, and the survival rate of disease mice is further prolonged (Nature Medicine,2017,23 (12): 1481-1487 circulation,2018,137 (24): 2613-2634). Accordingly, the present inventors evaluated the potential therapeutic effect of compound SN-011 in a cerebral ischemia model in rats. The experimental results show that the cerebral infarction area of the rats in the model group is remarkably increased after the rats are subjected to middle artery occlusion (MCAO) for 24 hours, and the cerebral infarction area can be remarkably improved by administering SN-011 at a low dose (1 mg/kg) and at a high dose (3 mg/kg) (figure 15A). The motor abilities of rats were evaluated 6 hours and 24 hours after the operation, and SN-011 administered was able to significantly improve the motor dysfunction of rats after stroke (FIGS. 15B to 15C). Further examining the expression of inflammatory genes in the cerebral cortex tissue of rats, it was found that the expression of Ifnb, ifna4, cxcl10, mcp-1, tnf-a and Il-6 genes in the cerebral cortex was significantly increased after the blocking of the middle artery in rats, and compared with that, SN-011 could significantly inhibit the expression of the above genes in the cerebral cortex tissue induced by ischemic injury (FIGS. 15D to 15I). The research results show that the compound SN-011 can obviously improve the damage of parenchymal organs induced by ischemia. Further, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006), and the compound of formula V (SN-010) all have similar effects to the compound of formula I (SN-011).

This suggests that the compounds of formula I-V of the present invention can be used for preparing drugs for preventing or treating STING-mediated ischemic cardiovascular and cerebrovascular diseases.

Example 15

SN-011 (compound of formula I) improves the cerebral ischemia injury of mice

Cerebral apoplexy can rapidly lead to the activation of microglia and the infiltration of peripheral immune cells, and the content of DNA in cerebrospinal fluid of a patient with the cerebral apoplexy is obviously increased. Recent studies have shown that the cGAS-STING pathway is activated after stroke and mediates post-stroke injury (EMBO Molecular Medicine,2020, 12 (4)). The experimental results show that: mice were injected intraperitoneally with a low dose of SN-011 (1 mg/kg) and a high dose of SN-011 (2 mg/kg) immediately after middle artery occlusion, and after 24 hours, compound SN-011 significantly inhibited the expression of Mcp-1, il-6 and Tnf-alpha genes (FIG. 16A); mice were injected intraperitoneally with a low dose of SN-011 (1 mg/kg) and a high dose of SN-011 (2 mg/kg) immediately after middle artery occlusion, and after 72 hours, compound SN-011 significantly inhibited the expression of Mcp-1, il-6 and Cxcl10 genes (FIG. 16B); the SN-011 (2 mg/kg) is injected into the abdominal cavity immediately after or 24 hours after the middle cerebral artery of the mouse is blocked, the SN-011 is continuously administrated to the seventh day after the stroke, and the movement coordination of the stroke mouse can be obviously improved by the compound SN-011 which is administrated immediately or after being delayed for 24 hours (figure 16C); immediately after the middle cerebral artery of the mouse is blocked or 24 hours later, SN-011 (2 mg/kg) is injected intraperitoneally and is continuously administrated to the seventh day after the stroke, and the movement coordination of the stroke mouse can be remarkably improved by immediately administrating the SN-011 or delaying administrating the SN-011 for 24 hours (figure 16D);

example 16

SN-011 (compound of formula I) ameliorates high fat diet-induced liver injury in mice and lipid-aggregated non-alcoholic fatty liver disease (NAFLD) is mainly characterized by hepatic steaggregation-induced hepatic steatosis, and when not effectively controlled, the excessively aggregated fat induces inflammatory injury and fibrosis of the liver and the disease progresses to non-alcoholic steatohepatitis (NASH). Epidemiological studies have shown that NASH is the most common factor leading to cirrhosis and liver carcinogenesis, and there is no currently effective drug for treating NASH. Recent studies found that expression of STING was significantly elevated in the liver of NAFLD patients, mainly concentrated in non-parenchymal cells of the liver such as macrophages and kupffer cells, and that knocking out expression of bone marrow cell STING protein in animal models could significantly improve high fat diet-induced NAFLD disease progression in mice (Gastroenterology, 2018,155 (6): 1971-1984. Accordingly, the present inventors evaluated the efficacy of SN-011 in a high fat diet-induced NAFLD model. After feeding C57BL6J male mice with 4-6 weeks of age with High Fat Diet (HFD), the intervention is carried out by starting to administer SN-011 with low dose (1 mg/kg) and high dose (2 mg/kg) 10 weeks later, and the biochemical indexes of the serum of the mice are detected 10 weeks later after continuous administration. The results showed that the serum levels of ALT, AST, TC and TG were significantly increased in HFD mice compared to normal diet fed mice, and the above-mentioned markers were significantly decreased in the mice serum after administration, indicating that SN-011 could ameliorate HFD diet-induced liver damage and reduce serum Total Cholesterol (TC) and Triglyceride (TG) levels (FIGS. 17A-17D). SN-011 administered significantly reduced body weight, liver weight and perirenal visceral fat weight in mice (FIGS. 17E-17G), and total cholesterol and triglyceride levels in the liver were also significantly reduced after administration (FIGS. 17H-17I). The above results confirm the regulation of lipid metabolism in mice by SN-011. Further examination of STING-activated immune and inflammatory cytokines and expression levels of regulatory lipid metabolism protein genes in the liver revealed that high fat diet-induced upregulation of liver Ifnb, cxcl10, mcp-1 and Tnfa genes was significantly improved after SN-011 administration (fig. 17J-17M), suggesting that SN-011 can improve liver inflammatory response and also significantly inhibit the upregulation of HFD-induced hepatic lipid synthesis-regulated protein Fatty Acid Synthase (FAS) and sterol regulatory element binding protein (SREBP-1 c) genes (fig. 17N-17O). The liver of the experimental group of mice was further pathologically sectioned to evaluate the inflammatory injury and lipid vacuole of the tissue, and the results showed that SN-011 can significantly improve inflammatory cell infiltration and lipid aggregation-induced bullous steatosis in the liver tissue of HFD mice (fig. 17P). Further, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006), and the compound of formula V (SN-010) all have similar effects to the compound of formula I (SN-011).

The results indicate that the compounds shown in the formulas I to V can be used for preparing the medicine for preventing or treating the non-alcoholic fatty liver disease (including NASH) mediated by STING.

Example 17

SN-011 (compound of formula I) ameliorates imiquimod-induced psoriasis-like inflammation an imiquimod-induced psoriasis-like inflammation model was established with BALB/c mice and it was observed whether compound SN-011 can reverse imiquimod-induced psoriasis-like inflammation. BALB/c mice were randomly divided into a blank Control group (Control group), a Model Control group (Model group), an SN-011 mg/kg group, and an SN-011 mg/kg group. Psoriasis skin lesion area and disease severity (PASI) score were used to observe disease progression and measure right ear thickness. H & E staining was performed to observe the thickness of the epidermis of the right ear and back skin and the condition of Munro micro-abscesses in each group of mice. The experimental results show (FIGS. 18 and 19) that the ear thickness of the Model group mice is significantly higher than that of the Control group mice, and the ear thickness of the compound SN-011 group mice is lower than that of the Model group mice, and the ear thicknesses are significantly different. The H & E staining results show (fig. 20) that compound SN-011 reduces mouse acanthoderma thickness with significant differences. Further, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006), and the compound of formula V (SN-010) all have similar effects to the compound of formula I (SN-011).

The results indicate that the compound can improve psoriasis-like inflammation and can be used for preparing a medicament for preventing or treating STING-mediated psoriasis.

In conclusion, the compounds of formulae I-V of the present invention or pharmaceutically acceptable salts or solvates thereof can be used in the preparation of a medicament for preventing or treating STING-mediated diseases.

The mechanism of action of the compounds of the invention is shown in figure 21. Taking the compound SN-011 (compound of formula I) as an example, under normal conditions, abnormal DNA in cytoplasm is recognized by cGAS to generate 2'3' -cGAMP molecules, 2'3' -cGAMP is then combined with endoplasmic reticulum regulatory protein STING to induce the conformation change of the STING protein to generate polymerization activation, then the polymerized STING protein is transferred to Golgi to recruit downstream proteins TBK1 and IRF3, TBK1 promotes IRF3 and NF-kappa B phosphorylation after autophosphorylation, and then the expression of nuclear-promoted interferon and inflammatory cytokines is entered; after the SN-011 molecule is added, the SN-011 molecule can be specifically combined into a pocket formed by the STING dimer, thereby preventing the CDNs molecule from activating the STING protein and blocking the transmission of signals.

Example 18

Tablet formulation

The compound of formula I prepared in example 1 (50 g), hydroxypropylmethylcellulose E (150 g), starch (200 g), appropriate amount of povidone K30 and magnesium stearate (1 g) were mixed, granulated and tabletted.

In addition, the compounds of formulae I to V of the present invention can be formulated into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches, or the like by adding different pharmaceutical excipients according to the conventional formulation method of pharmacopoeia 2015 edition.

Claims

1. The application of the benzene sulfonamide compound shown as any one or more of the following formulas I-V or the pharmaceutically acceptable salt thereof in preparing the medicines for preventing or treating psoriasis, cerebral ischemia injury, non-alcoholic steatohepatitis or Aicardi-Gouti res syndrome:

。

2. the use according to claim 1, wherein the compound of formula I-V or the pharmaceutically acceptable salt thereof is a salt formed from a metal ion or a pharmaceutically acceptable amine, ammonium ion or choline.